Identification of a defence mechanism in vivo against the leakage of enterokinase into the blood.

1. The serum proteinase inhibitors alpha 1-antitrypsin, alpha 2-macroglobulin, inter-alpha-trypsin inhibitor and C1-esterase inhibitor were found not to affect the catalytic activity of human enterokinase, whereas bovine trypsin activity was modified essentially as expected. Enterokinase was also not inhibited by Trasylol (trypsin inhibitor from bovine lung) or bovine pancreatic trypsin inhibitor. No other component in human or mouse serum complexing with enterokinase was identified. 2. Human enterokinase administered intravenously into mice was rapidly cleared from the circulation with a half-life of 2.5 min. This removal was not the result of the difference in species, since partially purified mouse enterokinase was cleared at the same rate as the human enzyme. Clearance was mediated by recognition of the carbohydrate portion of enterokinase and not through specific recognition of its catalytic site. Immunofluorescent staining showed that the enzyme accumulated in the liver. Attempts to block the clearance by the simultaneous infusion of competing glycoproteins suggested that enterokinase was taken up by hepatocytes. Of the glycoproteins tested only two, human lactoferrin (terminal fucosyl alpha 1 leads to 3 N-acetylglucosamine) and bovine asialo-fetuin (terminal galactosyl beta 1 leads to 4 N-acetylglucosamine) were weakly competitive. Two inhibitors of endocytosis, Intralipid and Triton WR1339, failed to delay the removal of enterokinase. It is proposed that enterokinase is cleared from the circulation by an as yet uncharacterized hepatocyte receptor.     

Subcellular localization of enterokinase in human small intestine

Enterokinase (enteropeptidase, EC 3.4.4.8) was found to be purified to the same extent as sucrase and alkaline phophatase when human intestinal brush border membrane was isolated. It is concluded that, in man as in other mammals, enterokinase activity occurs in close association with the brush border membrane.However, a second localization was also found. A fraction of the mucosal homogenate containing only small amounts of brush border but large amounts of endoplasmic reticulum, basolateral membranes and mitochondria (Fraction P₁) contained a disproportionately high amount of enterokinase. The enzyme in this particulate fraction occurred in a not fully active form.     

重组牛肠激酶轻链基因在大肠杆菌中的表达与纯化

肠激酶（Enteroloinase,EK,EC3.4.21.9）是一种以异源二聚体形式存在于哺乳动物十二指肠内的丝氨酸蛋白酶,通过在位点（Asp）4-Lys的羧基端进行高效特异酶切,将胰蛋白酶原激活为胰蛋白酶。以GenBank公共数据库中牛肠激酶轻链基因序列（Accession No.NM174439）设计引物,利用RT-PCR方法合成牛肠激酶轻链基因片段,并克隆进pET39b载体中DsbA片段的C’端,转化大肠杆菌BL21（DE3）,获得DsbA/牛肠激酶轻链融合蛋白,经镍离子螯合层析纯化,每升培养液中可得到2.7-3.0mg重组牛肠激酶,对含有肠激酶酶切位点的IL-11/MBP融合蛋白进行酶切,结果表明,酶解率可达到95%以上,为重组牛肠激酶的大规模生产打下了基础。     

Occurrence and Heterogeneity of Chymotrypsin Inhibitors in Vegetative Tissues of Barley

Inhibitors of chymotrypsin and the alkaline proteinase of Aspergillus oryzae were present in the shoots of barley seedlings and weak activities were also detected in the shoot tops of 6-week-old plants. Treatments which induce inhibitor formation in tomato and potato leaves had no effect when tested on mature leaves, seedlings, or young tillers of barley. Fractionation experiments with isoelectric focusing showed that the barley leaves contained several proteinase inhibitors acting on both chymotrypsin and the Aspergillus proteinase, and one inhibitor which acted only on the Aspergillus enzyme. All of these inhibitors were different from the five Aspergillus proteinase inhibitors which are abundant in the endosperm of resting seeds. Two chymotrypsin inhibitors with weaker activity on the Aspergillus proteinase were present in rootlets and also in embryos of resting seeds. These inhibitors were different from both the endospermal inhibitors and the inhibitors present in young leaves.     

Studies on the activation of canine trypsinogens in vitro.

The activation of canine anionic and cationic trypsinogen by enterokinase, trypsin, thrombin, plasmin and extracts from canine granulocytes were studied in vitro. Enterokinase activates both trypsinogens about 1000 times faster than trypsin. The enterokinase-catalyzed activation is not inhibited by the main serum protease inhibitors, alpha-macroglobulin and alpha 1-antitrypsin. alpha-Macroglobulin cannot inhibit the activation of the trypsinogens by trypsin but this reaction is completely inhibited by alpha 1-antitrypsin. The results are discussed in relation to the pathogenesis of acute pancreatitis.     

Receptor-mediated endocytosis of enterokinase by rat liver. Preliminary characterisation of low-density endosomes

The endocytosis of enterokinase by rat hepatocytes has been studied both in a perfused liver system and in the intact, anaesthetised animal. 10 min after administration of the enzyme, only 70% of the activity was cleared by the perfused liver, whereas clearance was total in the intact animal. In both cases, about 85% of the internalised enzyme co-purified with the smooth microsomes and virtually all (more than 90%) of the catalytic activity was latent and could only be detected in the presence of detergent. After 10 min, 22.5% of the activity remained with the sinusoidal plasma membrane in the case of the perfused liver, while for the intact animal this figure was only 10%, confirming the more efficient clearance of enterokinase in the intact animal. Further subcellular fractionation showed that in the anaesthetised animal 8% of the internalised enzyme was associated with a low-density Golgi-like endosomal compartment (prepared from the mitochondrial pellet), whereas the corresponding value for the perfused liver was only 2.5%. Enterokinase specific activity was also up to 50-times greater in the low-density endosomes prepared from the intact animal. A second low-density Golgi-like compartment (purified from the smooth microsomes) also contained latent enterokinase, which together with the endosomes derived from the mitochondria accounted for 20% of the total enterokinase internalised by the liver 10 min after its administration to the intact animal. The passage of enterokinase through these two low-density compartments was shown not to be synchronous with its passage through the peripheral (sinusoidal membrane) and internal endosomes (smooth microsomes). There were qualitative differences in marker enzymes and polypeptide composition between the mitochondria and microsome-derived low-density endosomes. The sub-fractionation of low-density fractions on shallow sucrose gradients revealed a complex enzyme and polypeptide heterogeneity both between and within fractions. There was an apparent density-dependent separation of enterokinase from galactosyltransferase and the asialoglycoprotein receptor which was coincident with marked changes in the polypeptide composition of the endosomal membranes, particularly in the 30-45 kDa range.     

The effect of limited proteolysis on rabbit muscle creatine kinase.

We report a novel assay method for enterokinase capable of detecting approx. 1 fmol of enzyme. The method depends on quantification of the release of specifically radiolabelled activation peptides from bovine trypsinogen and is unaffected by trypsin inhibitors. The assay is applicable to biological fluids such as serum. The substrate was produced by selective epsilon-amidination of bovine trypsinogen followed by acetylation with [3H]acetic anhydride and deprotection. The assay has been used to study the effects of pH, Ca2+, ionic strength abd glycodeoxycholate on enterokinase activity.     

Receptor-mediated endocytosis of enterokinase by rat liver: Preliminary characterisation of low-density endosomes

The endocytosis of enterokinase by rat hepatocytes has been studied both in a perfused liver system and in the intact, anaesthetised animal. 10 min after administration of the enzyme, only 70% of the activity was cleared by the perfused liver, whereas clearance was total in the intact animal. In both cases, about 85% of the internalised enzyme co-purified with the smooth microsomes and virtually all (more than 90%) of the catalytic activity was latent and could only be detected in the presence of detergent. After 10 min, 22.5% of the activity remained with the sinusoidal plasma membrane in the case of the perfused liver, while for the intact animal this figure was only 10%, confirming the more efficient clearance of enterokinase in the intact animal. Further subcellular fractionation showed that in the anaesthetised animal 8% of the internalised enzyme was associated with a low-density Golgi-like endosomal compartment (prepared from the mitochondrial pellet), whereas the corresponding value for the perfused liver was only 2.5%. Enterokinase specific activity was also up to 50-times greater in the low-density endosomes prepared from the intact animal. A second low-density Golgi-like compartment (purified from the smooth microsomes) also contained latent enterokinase, which together with the endosomes derived from the mitochondria accounted for 20% of the total enterokinase internalised by the liver 10 min after its administration to the intact animal. The passage of enterokinase through these two low-density compartments was shown not to be synchronous with its passage through the peripheral (sinusoidal membrane) and internal endosomes (smooth microsomes). There were qualitative differences in marker enzymes and polypeptide composition between the mitochondria and microsome-derived low-density endosomes. The sub-fractionation of low-density fractions on shallow sucrose gradients revealed a complex enzyme and polypeptide heterogeneity both between and within fractions. There was an apparent density-dependent separation of enterokinase from galactosyltransferase and the asialoglycoprotein receptor which was coincident with marked changes in the polypeptide composition of the endosomal membranes, particularly in the 30–45 kDa range.     

Do-it-yourself histidine-tagged bovine enterokinase: A handy member of the protein engineer's toolbox

Enterokinase, a two-chain duodenal serine protease, activates trypsinogen by removing its N-terminal propeptide. Due to a clean cut after the non-primed site recognition sequence, the enterokinase light chain is frequently employed in biotechnology to separate N-terminal affinity tags from target proteins with authentic N-termini. In order to obtain large quantities of this protease, we adapted an in vitro folding protocol for a pentahistidine-tagged triple mutant of the bovine enterokinase light chain. The purified, highly active enzyme successfully processed recombinant target proteins, while the pentahistidine-tag facilitated post-cleavage removal. Hence, we conclude that producing enterokinase in one's own laboratory is an efficient alternative to the commercial enzyme.     

Incorporation of bovine enterokinase in reconstituted soybean phospholipid vesicles

Bovine enterokinase was incorporated into vesicles reconstituted from a soybean phospholipid mixture. A thin film hydration procedure (MacDonald, R. I., and MacDonald, R. C. (1975) J. Biol. Chem. 250, 9206-9214) produced vesicles with 40% of the enterokinase activity bound in the membrane. The highest incorporation was observed when cholesterol or dimyristoylphosphatidylethanolamine was added to the soybean phospholipids. Crude and highly purified enterokinase preparations were incorporated to the same extent suggesting that other membrane components were not required for a successful reconstitution. The properties of enterokinase in phospholipid vesicles were compared with those of alkaline phosphatase, which was also added to the reconstitution system, and with the enzyme activities present in vesicles prepared from brush-border membranes. The enzyme activities were not released by solutions of high ionic strength and remained associated with the phospholipid vesicles on gel filtration, ultracentrifugation, and sucrose density centrifugation. Enterokinase and alkaline phosphatase had their active sites exposed to substrate in the brush-border membrane vesicles. In soybean phospholipid vesicles half of the active sites of both enzymes were on the outside, since release of the enzyme with Triton X-100 almost doubled the units of enzyme present. Incubation of the soybean phospholipid and brush-border membrane vesicles with papain released the exposed molecules of enterokinase. The released enzyme molecules were fully active but could not be reincorporated into phospholipid vesicles. This suggests that the structure imbedded in the lipid bilayer was essential for a successful reconstitution. We conclude that the reconstituted soybean phospholipid vesicles are a suitable membrane system for the further study of membrane-bound enterokinase.     

Expression,purification, and characterization of a biologically active bovine enterokinase catalytic subunit in Escherichia coli

Enterokinase (EC 3.4.21.9) is a serine proteinase in the duodenum that exhibits specificity for the sequence (Asp)(4)-Lys. It converts trypsinogen to trypsin. Its high specificity for the recognition site makes enterokinase (EK) a useful tool for in vitro cleavage of fusion proteins. cDNA encoding the catalytic chain of Chinese bovine enterokinase was cloned and its encoding amino acid sequence is identical to the previously reported sequence although there are two one-base mutations which do not change the encoded amino acid. The EK catalytic subunit cDNA was cloned into plasmid pET32a, and fused downstream to the fusion partner thioredoxin (Trx) and the following DDDDK enterokinase recognition sequence. The recombinant bovine enterokinase catalytic subunit was expressed in Escherichia coli BL21(DE3), and most products existed in soluble form. After an in vivo autocatalytic cleavage of the recombinant Trx-EK catalytic domain fusion protein, intact, biologically active EK catalytic subunit was released from the fusion protein. The recombinant intact EK catalytic subunit was purified to homogeneity with a specific activity of 720 AUs/mg protein through ammonium sulfate precipitation, DEAE chromatography, and gel filtration. The purified intact EK catalytic subunit has a K(m) of 0.17 mM, and K(cat) is 20.8s(-1). From 100 ml flask culture, 4.3 mg pure active EK catalytic subunits were obtained.     

Optimization of the fermentation and downstream processes for human enterokinase production in Pichia pastoris

Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml. Lower cultivation temperature had a negative impact on the enzyme accumulation during this fermentation strategy. Downstream processes were focused on buffer environment optimization directly after cultivation, as at this time, the most amount of the activity is eliminated by endogenous proteases. Slightly positive effect on enzyme activity in the medium had an addition of liquid storage solution of EDTA and KOH to adjust pH to 8 and molarity of the EDTA to 50 mM. During the purification process, a significant amount of the enzyme was detected to be lost, which counted up to 90%. The purified enzyme, enterokinase, kept quality standard of the published enzymes.

     

Isolation and characterization of the proteinase inhibitor-inducing factor from tomato leaves. Identity and activity of poly- and oligogalacturonide fragments

Mild acid hydrolysis of a small (Mr = 6 kDa) pectic polysaccharide isolated from tomato leaves, an inducer of the synthesis and accumulation of two proteinase inhibitors in excised tomato plants, yielded a alpha-D-polygalacturonic acid polymer with degree of polymerization = 20 that retained proteinase inhibitor-inducing activity. Enzymic and acid hydrolysis of this polygalacturonan yielded a series of alpha-1,4-D-galacturonic acid oligomers with degrees of polymerization from 2 to 6 which were purified to homogeneity and assayed for proteinase inhibitor-inducing activity in young excised tomato plants. All of the oligomers exhibited activity. The hexagalacturonide possessed the highest activity and the trimer the lowest. The evidence supports a possible role for plant cell wall fragments as systemic messengers that regulate the expression of proteinase inhibitor genes in plant leaves in response to pest attacks.     

Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression

To assess the utility of the tomato fruit-specific E8 gene's promoter for driving vaccine antigen expression in plant, the 2.2 kb and 1.1 kb E8 promoters were isolated and sequenced from Lycopersicon esculentum cv. Jinfeng #1. The 1.1 kb promoter was fused to vaccine antigen HBsAg M gene for the transfer to Nicotiana tabacum, and the CaMV 35S promoter was used for comparison. Cholera toxin B (ctb) gene under the control of the 1.1 kb promoter was transformed into both N. tabacum and L. esculentum. Southern blot hybridization confirmed the stable integration of the target genes into the tomato and tobacco genomes. ELISA assay showed that the expression product of HBsAg M gene under the control of the 1.1 kb E8 promoter could not be detected in transgenic tobacco tissues such as leaves, flowers, and seeds. In contrast, the expression of HBsAg M gene driven by CaMV 35S promoter could be detected in transgenic tobacco. ELISA assay for CTB proved that the 1.1 kb E8 promoter was able to direct the expression of exotic gene in ripe fruits of transgenic tomato, but expression was absent in leaf, flower, and unripe fruit of tomato, and CTB protein was not detected in transgenic tobacco tissues such as leaves, flowers, and seeds when the gene was under the control of the 1.1 kb E8 promoter. The results indicated that the E8 promoter acted not only in an organ-specific, but also in a species-specific fashion in plant transformation.     

LE-ACS6启动子在LE-ACS6::GUS转基因拟南芥中的特异性

通过对不同发育时期LE-ACS6::GUS转基因拟南芥中GUS表达特异性的研究,证明LE-ACS6基因的启动子在拟南芥中也表现启动参与第一系统乙烯合成的关键酶基因的活性.在转基因拟南芥中,LE-ACS6启动子还表现响应外源生长素处理、伤害处理等多种刺激因子的特点.     

The physiological implications of urease inhibitors on N metabolism during germination of Pisum sativum and Spinacea oleracea seeds

The development of new nitrogen fertilizers is necessary to optimize crop production whilst improving the environmental aspects arising from the use of nitrogenous fertilization as a cultural practice. The use of urease inhibitors aims to improve the efficiency of urea as a nitrogen fertilizer by preventing its loss from the soil as ammonia. However, although the action of urease inhibitors is aimed at the urease activity in soil, their availability for the plant may affect its urease activity. The aim of this work was therefore to evaluate the effect of two urease inhibitors, namely acetohydroxamic acid (AHA) and N-(n-butyl) thiophosphoric triamide (NBPT), on the germination of pea and spinach seeds. The results obtained show that urease inhibitors do not affect the germination process to any significant degree, with the only process affected being imbibition in spinach, thus also suggesting different urease activities for both plants. Our findings therefore suggest an activity other than the previously reported urolytic activity for urease in spinach. Furthermore, of the two inhibitors tested, NBPT was found to be the most effective at inhibiting urease activity, especially in pea seedlings.     

Endo-[beta]-Mannanase Activity from Individual Tomato Endosperm Caps and Radicle Tips in Relation to Germination Rates

Endo-[beta]-mannanase is hypothesized to be a rate-limiting enzyme in endosperm weakening, which is a prerequisite for radicle emergence from tomato (Lycopersicon esculentum Mill.) seeds. Using a sensitive, single-seed assay, we have measured mannanase activity diffusing from excised tomato endosperm caps following treatments that alter the rate or percentage of radicle emergence. Most striking was the 100- to more than 10,000-fold range of mannanase activity detected among individual seeds of highly inbred tomato lines, which would not be detected in pooled samples. In some cases a threshold-type relationship between mannanase activity and radicle emergence was observed. However, when radicle emergence was delayed or prevented by osmoticum or abscisic acid, the initial increase in mannanase activity was unaffected or even enhanced. Partially dormant seed lots displayed a bimodal distribution of activity, with low activity apparently associated with dormant seeds in the population. Gibberellin- and abscisic acid-deficient mutant seeds exhibited a wide range of mannanase activity, consistent with their variation in hormonal sensitivity. Although the presence of mannanase activity in the endosperm cap is consistently associated with radicle emergence, it is not the sole or limiting factor under all conditions.     

Intracellular co-localization of trypsin-2 and matrix metalloprotease-9: possible proteolytic cascade of trypsin-2, MMP-9 and enterokinase in carcinoma

Tumor-associated trypsin-2 and matrix metalloprotease-9 (MMP-9) are associated with cancer, particularly with invasive squamous cell carcinomas. They require activation for catalytical competence via proteolytic cascades. One cascade is formed by enterokinase, trypsin-2 and MMP-9; enterokinase activates trypsinogen-2 to trypsin-2, which is an efficient proMMP-9 activator. We describe here that oral squamous cell carcinomas express all members of this cascade: MMP-9, trypsin-2 and enterokinase. The expression of enterokinase in a carcinoma cell line not derived from the duodenum was shown here for the first time. Enterokinase directly cleaved proMMP-9 at the Lys65-Ser66 site, but failed to activate it in vitro. We demonstrated by confocal microscopy that MMP-9 and trypsin-2 co-localized in intracellular vesicles of the carcinoma cells. This co-localization of trypsin-2 and MMP-9 resulted in intracellular proMMP-9 processing that represented fully or partially activated MMP-9. However, although both proteases were present also in various bone tumor tissues, MMP-9 and trypsin-2 never co-localized at the cellular level in these tissues. This suggests that the intracellular vesicular co-localization, storage and possible activation of these proteases may be a unique feature for aggressive epithelial tumors, such as squamous cell carcinomas, but not for tumors of mesenchymal origin.     

An immunochemical study of antigen expression in potato spindle tuber viroid (PSTV) - infected tomato leaves and calluses

A polyspecific antiserum against protein extracted from PSTV-infected tomato leaves was prepared and the IgGs were separated by affinity chromatography on a beaded cellulose adsorbent with an immobilized “healthy” antigen. The antibody not adsorbed entered into a preferential reaction with the antigen from PSTV-infected leaves as estimated by an enzyme-linked immunosorbent assay. The immunochemical reactions did not significantly exceed the control background, if antigens from tomato leaves infected with potato viruses X, Y and M were analyzed. By immunoblot technique we revealed, however, that several antigens not detected in healthy leaves appeared in the leaves infected either with PSTV or with viruses X and M. An accumulation of a major antigen having a molecular mass of about 70 kDa was observed in viroid-infected leaves only, suggesting the specificity for viroid infection. The antigen was found not to be an alkaline endoproteinase - the pathogenesis-related protein P-69. Some antigens with molecular masses approximately 38.0, 23.7 and 22 kDa, which occurred in PSTV-infected leaves and in healthy calluses, were not detectable in PSTV-infected calluses. No reaction exceeding the control level was observed using enzyme-linked immunosorbent assay for antigens from silver nitrate-treated tomato leaves, although such leaves showed symptoms similar to that caused by viroids.     

Integrated bioprocessing in Saccharomyces cerevisiae using green fluorescent protein as a fusion partner

In this study, we examine the use of green fluorescent protein (GFP) for monitoring a hexokinase (HXK)-GFP fusion protein in Saccharomyces cerevisiae for various events including expression, degradation, purification, and localization. The fusion, HXK-EK-GFP-6 x His, was constructed where the histidine tag (6 x His) would allow for convenient affinity purification, and the enterokinase (EK) cleavage site would be used for separation of HXK from GFP after affinity purification. Our results showed that both HXK and GFP remained active in the fusion and, more importantly, that there was a linear correlation between HXK activity and GFP fluorescence. Enterokinase cleavage studies revealed that both GFP fluorescence intensity and HXK activity remained unchanged after separation of the fusion proteins, which indicated that fusion of GFP did not cause structural alteration of HXK and thus did not affect the enzymatic activity of HXK. We also found that degradation of the fusion protein occurred, and that degradation was limited to HXK with GFP remaining intact in the fusion. Confocal microscopy studies showed that while GFP was distributed evenly in the yeast cytosol, HXK-GFP fusion followed the correct localization of HXK, which resulted in a di-localization of both cytosol and the nucleus. GFP proved to be a useful fusion partner that may lead to the possibility of integrating the bioprocesses by quantitatively following the entire process visually.