Calcium-Activated Neutral Proteinase of Human Brain: Subunit Structure and Enzymatic Properties of Multiple Molecular Forms

Abstract Calcium-activated neutral proteinase (CANP) was purified 2,625-fold from postmortem human cerebral cortex by a procedure involving chromatography on diethylaminoethyl (DEAE)-cellulose, phenyl-Sepharose, Ultrogel AcA-44, and DEAE-Biogel A. The major active form of CANP exhibited a molecular weight of 94–100 kilodaltons (Kd) by gel filtration on Sephacryl 300 and consisted of 78-Kd and 27-Kd subunits. Two-dimensional gel electrophoresis resolved the small subunit into two molecular species with different isoelectric points. CANP degraded most human cytoskeletal proteins but was particularly active toward fodrin and the neurofilament protein subunits (145 Kd > 200 Kd > 70 Kd). The enzyme required 175 μMCa²⁺ for half-maximal activation and 2 mM Ca²⁺ for optimal activity toward [methl-¹⁴C]azocasein. Other divalent metal ions were poor activators of the enzyme, and some, including copper, lead, and zinc, strongly inhibited the enzyme. Aluminum, a neurotoxic ion that induces neurofilament accumulations in mammalian brain, inhibited the enzyme 47% at 1 mM and 100% at 5 mM A second CANP form lacking the 27-Kd subunit was partially resolved from the 100-Kd heterodimer during DEAE-Biogel A chromatography. The 78-Kd monomer exhibited the same specific activity, calcium ion requirement, pH optimum, and specificity for cytoskeletal proteins as the 100-Kd heterodimer, suggesting that the 27-Kd subunit is not essential for the major catalytic properties of the enzyme. The rapid autolysis of the 27-Kd subunit to a 18-Kd intermediate when CANP is exposed to calcium may explain differences between our results and previous reports, which describe brain mCANP in other species as a 76-80-Kd monomer or a heterodimer containing 76-80-Kd and 17-20-Kd subunits. The similarity of the 100-Kd human brain CANP to CANPs in nonneural tissues indicates that the heterodimeric form is relatively conserved among various tissues and species.     

枯草杆菌中性蛋白酶天然抑制剂的筛选

用透明因法和福林──酚法筛选了２１种植物中的蛋白酶活性抑制剂,其中来自鼓皮、高粱、玉米和土豆的抑制剂的活性在本研究条件下达５０％以上。进一步研究了麸皮、玉米两种抑制剂在不同温度和ｐＨ值下的稳定性,结果表明,两者的稳定性相差甚远,是两类不同的抑制剂。玉米抑制剂比麸皮抑制剂更稳定。     

昆虫消化酶抑制剂与害虫防治

生物源昆虫消化酶抑制剂主要包括蛋白酶抑制剂和淀粉酶抑制剂。昆虫消化酶抑制剂能通过降低或抑制昆虫蛋白酶或淀粉酶的活性,而影响昆虫的正常生长发育,使其生长缓慢,虚弱,甚至导致死亡。本文就生物源昆虫消化酶抑制剂对昆虫生化、生理代谢和生长发育的影响,消化酶抑制剂的作用机理,植物中昆虫消化酶抑制剂的诱导产生等进行了介绍。同时,探讨了生物源蛋白酶和淀粉酶抑制剂在害虫防治中的应用前景。     

Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds

Dry mature seeds of winged bean (Psophocarpus tetragonolobus L., DC.) (WB) contain several proteinase inhibitors. Two-dimensional gel analysis of WB seed protein followed by activity visualization using a gel-X-ray film contact print technique revealed at least 14 trypsin inhibitors (TIs) in the range of 28-6 kD. A total of seven inhibitors (WBTI-1 to 7) were purified by heat treatment and gel filtration followed by elution from preparative native gels. Based on their biochemical characterization such as molecular mass, pI, heat stability, and susceptibility to inactivation by reducing agents, WBTI-1 to 4 are Kunitz type inhibitors while WBTI-5 to 7 are classified as Bowman-Birk type serine proteinase inhibitors. Although Kunitz type TIs (20-24 kD) of WB have been reported, the smaller TIs that belong to the Bowman-Birk type have not been previously characterized. Seven major TIs isolated from WB seed were individually assessed for their potential to inhibit the gut proteinases (HGP) of Helicoverpa armigera, a pest of several economically important crops, which produces at least six major and several minor trypsin/chymotrypsin/elastase-like serine proteinases in the gut. WBTI-1 (28 kD) was identified as a potent inhibitor of HGP relative to trypsin and among the other WBTIs; it inhibited 94% of HGP activity while at the same concentration it inhibited only 22% of trypsin activity. WBTI-2 (24 kD) and WBTI-4 (20 kD) inhibited HGP activity greater than 85%. WBTI-3,-5,-6 and-7 showed limited inhibition of HGP as compared with trypsin. These results indicate that WBTIs have different binding potentials towards HGP although most of the HGP activity is trypsin-like. We also developed a simple and versatile method for identifying and purifying proteinase inhibitors after two-dimensional separation using the gel-X-ray film contact print technique.     

High molecular mass kininogen inhibits metalloproteinases of Bothrops jararaca snake venom

High molecular mass kininogen (HK) purified from Bothrops jararaca (Bj) plasma was tested on activities of the Bj venom in vivo and in vitro. Results showed that, when incubated with BjHK, the Bj venom presented inhibition on hemorrhagic, edema forming, myotoxic, and coagulant activities. It is well known that metalloproteinases are directly or indirectly involved in these activities. Similarly, human HK inhibits the hemorrhagic effect of the Bj venom as well as hemorrhagic and enzymatic effects of jararhagin, a hemorrhagic metalloproteinase isolated from Bj venom. Complex between HK and jararhagin was not detected by gel filtration. Nevertheless, the inhibitory effect of the hemorrhagic activity of the venom was only partial when HK was pre-incubated with 0.4mM ZnCl(2) or with 0.45mM CaCl(2). These data suggest that the inhibitory effect depends, at least partially, on the competition for ions between kininogen and metalloproteinases of the venom.     

Bowman-birk proteinase inhibitor confers heavy metal and multiple drug tolerance in yeast

Cultured Coptis japonica cells show tolerance to various toxic compounds. By yeast functional screening of cadmium (Cd) plates with its cDNA library, we isolated a gene encoding Bowman-Birk proteinase inhibitor (CjBBI). The yeast transformant of CjBBI showed multiple tolerance to various drugs adding to Cd, and revealed reduced Cd accumulation in cells. Preferential organs for Cjbbi expression were aerial parts of intact plants, and the subcellular localization of CjBBI was shown, using its green fluorescent protein fusion, to be the apoplast. Induction of Cjbbi expression by Cd treatment suggested that CjBBI was responsible for the tolerance to Cd observed in C. japonica cells.     

Molecular Characterization of a Novel Bacteriocin and an Unusually Large Aggregation Factor of <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> subsp. <Emphasis Type="Italic">paracasei</Emphasis> BGSJ2-8, a Natural Isolate from Homemade Cheese

Screening the collection of natural isolates from semi-hard homemade cheese resulted in isolation and characterization of strain Lactobacillus paracasei subsp. paracasei BGSJ2-8. The strain BGSJ2-8 harbors several important phenotypes, such as bacteriocin production, aggregation phenomenon, and production of proteinase. Bacteriocin SJ was purified by three-step chromatography. Mass spectrometry established molecular mass of the active peptide at 5372 Da. The auto-aggregation phenotype of wild-type (WT) strain was mediated by secreted aggregation-promoting factor (protein of molecular mass > 200 kDa), probably acting in cooperation with other cell surface protein(s). Comparative study of WT and its spontaneous nonaggregating derivative revealed that aggregation factor was responsible for the observed differences in the bacteriocin and proteinase activities. Bacteriocin SJ activity and resistance to different stresses were higher in the presence of aggregating factor. In contrast, proteinase activity was stronger in the nonaggregating derivative.     

A colorimetric-based amplification system for proteinases including MMP2 and ADAM8

We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads. The amount of product is then quantified using a standard HRP color development assay employing 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂). This HRP amplification system represents a new approach to proteinase assays and could be applied to other enzymes, such as lipases, esterases, and kinases, as long as the unreacted substrate can be physically separated from the product and catalysis by the enzyme to be quantified is not impaired dramatically by steric hindrance from the HRP entity.     

胰蛋白酶诱导人脐静脉内皮细胞分泌白细胞介素-8

目的:研究胰蛋白酶对IL-8释放的影响。方法:分离、培养人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)、倒置显微镜观察形态变化,流式细胞术检测内皮细胞标志和蛋白酶活化受体-2(proteinase-activated receptor-2,PAR-2)表达,ELISA检测HUVECs培养上清中IL-8水平。结果:HUVECs表达内皮细胞标志和PAR-2。刺激16 h,1 g/ml胰蛋白酶和100M PAR-2激活肽组HUVECs单层均匀性降低。胰蛋白酶能够显著刺激HUVECs释放IL-8,PAR-2激活肽也诱导IL-8水平升高。蛋白酶抑制剂和PAR-2抑制肽均能够显著抑制胰蛋白酶诱导的IL-8释放。PAR-2激活肽和胰蛋白酶诱导升高的IL-8水平之间成正相关性。结论:胰蛋白酶很可能通过PAR-2激活促进血管内皮细胞释放IL-8。     

Intranasal immunisation with a 62 kDa proteinase combined with cholera toxin or CpG adjuvant protects against Trichomonas vaginalis genital tract infections in mice

Trichomonosis, caused by the protozoan parasite Trichomonas vaginalis, is one of the most frequent sexually transmitted diseases and is widely spread in all continents. Trichomonas vaginalis as well as other protozoan organisms have high levels of proteolitic activity mainly of the cysteine-proteinase type. This activity is necessary for recognition and adhesion of the parasite to the superficial epithelial cells of the host. In the present study, we show that intranasal immunisation with a 62 kDa cysteine-proteinase purified from T. vaginalis excretion-secretion products in combination with cholera toxin or with synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG motifs (CpG-ODN) elicits 62kDa specific IgG and IgA in vaginal lavage fluid and specific IgG in serum. This immunisation protocol resulted in enhanced elimination of parasites following intravaginal challenge of BALB/c mice.