海带,裙带菜和紫菜蛋白酶谱的研究

采用复性电泳方法对海带（ＬａｍｉｎａｒｉａＬａｍｘ．）、裙带菜（ＵｎｄａｒｉａＳｕｒｉｎｇａｒ）和紫菜（ＰｏｒｐｈｙｒａＣ．Ａｇ．）３个属１１种海藻的蛋白酶进行了研究,分析了海带、裙带菜和紫菜在不同ｐＨ条件下,蛋白酶种类的分布情况和活性差异。结果表明：（１）蛋白酶种类和活性受ｐＨ的影响和制约,其最适ｐＨ为８．０,此条件下蛋白酶的种类最多,活性最强;（２）亲缘关系越近,蛋白酶种类和活性越相似,海带同属不同种之间存在相同的蛋白酶,而不同属的海藻中很难找到相同的蛋白酶带;（３）海带属不同种所共有的两种蛋白酶（分子量１９ｋＤ和１８ｋＤ）是其特征性酶带;（４）蛋白酶谱可作为海藻分类依据之一。     

中国大鲵消化系统13种器官的蛋白水解酶种类和活性分析

蛋白水解对生命活动是必不可少的(Vassali et al., 1994),蛋白质的酶解修饰(Xu et al.,1999)、细胞的迁移、组织再生与修复、消化系统对食物中蛋白质的消化等均与蛋白水解酶有关(Baimbridge et al.,1992),许多病理过程也与蛋白水解酶功能失调有关(Teichert et al., 1989; Monard, 1988).因此开展大鲵消化系统各器官的蛋白水解酶种类和性质的研究,对了解大鲵消化系统各器官的功能、演化及大鲵的营养需求、食性、消化生理等是必要的.本文对大鲵消化系统各器官的蛋白水解酶特征进行了初步分析,现将结果报道如下.     

金鱼不同组织器官及胚胎发育不同时期蛋白水解酶的种类和活性变化

采用复性电泳方法研究了金鱼组织器官蛋白水解酶及个体发生过程中蛋白水解酶的种类和活性变化,主要结果表明：⑴金鱼各组织器官蛋白水解酶种类差异不大,大多数组织器官都具有１１３、６９、２０、１６ｋＤ四条带,但不同组织器官常具有其特异性蛋白水解酶;肠道蛋白水解酶种类最多、活性最强。⑵蛋白水解酶的活性受ｐＨ值影响和制约,大多数组织器官蛋白水解酶活性最适ｐＨ值为８．５。⑶在金鱼胚胎发育早期（从卵裂到心跳期）多数     

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS-PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to inhibit crude extract protease activity on Madin-Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.     

Protease activities in carp retina

Proteases of carp retina were examined by electrophoresis and fluorogenic assays. A 70 kD serine protease with an alkaline pH optimum was detected in gelatin-containing polyacrylamide gels. A similar enzyme was found in carp brain and muscle, but not in lens. Using aminomethylcoumarin (MCA) substrates, activities that hydrolysed Z-Phe-Arg-MCA, Boc-Ala-Gly-Pro-Arg-MCA and various aminoacyl-MCAs were detected. The Z-Phe-Arg-MCA hydrolase was an acidic cysteine protease, whereas the Boc-Ala-Gly-Pro-Arg-MCA hydrolase was an alkaline cysteine protease. All aminoacyl hydrolase activities tested were inhibited by bestatin and o-phenanthroline, but not by inhibitors of serine, cysteine and aspartic proteases, suggesting they are metalloaminopeptidases. Of the substrates tested, Tyr-MCA was the most readily hydrolysed aminoacyl substrate. Preliminary evidence was obtained suggesting that levels of these activities do not differ between light- and dark-adapted retinae.

The proteases have a potential involvement in retinal functioning and show similarities to other proteases known to act in the central nervous system. In particular, the Tyr-MCA hydrolase may be related to an enzyme known to remove the N-terminal tyrosine residue from enkephalin.     

CELL-ASSOCIATED PROTEOLYTIC ENZYMES FROM MARINE PHYTOPLANKTON1

Despite their central importance in cell metabolism, little is known about proteases in marine phytoplankton. We surveyed caseinolytic and leucine aminopeptidase (LAP) activities in log-phase cultures of the chlorophyte Dunaliella tertiolecta Butcher, the diatom Thalassiosira weissflogii (Gru.) Fryxell et Hasle, the chrysophyte Isochrysis galbana Parke, the coccolithophorid Emiliania huxleyi (Lohm.) Hay et Mohler, and the cyanobacterium Synechococcus sp. (WH 5701). LAP activity was very low at pH < 6 and peaked between pH 7.5 and 8.5 in all species, whereas caseinolytic activity in most species showed only minor peaks in the pH 4–5 range and broad maxima above pH 8. Thus, acidic vacuolar proteases apparently represented only a small fraction of total protease activity. Attempts to classify proteases using selective inhibitors were inconclusive. Neither the serine/cysteine protease inhibitor leupeptin nor the aspartic protease inhibitor pepstatin. A inhibited caseinolytic or LAP activity in any species. The metalloprotease inhibitor EDTA was only effective against LAP activity in some species, causing average decreases of 30–50%, whereas the cysteine/serine protease inhibitor phenyl methyl sulfonylfluoride achieved at best a 30–60% decrease in caseinolytic activity. Caseinolytic activities were remarkably stable. At pH 7.5 and 25°C, extracts of D. tertiolecta, E. huxleyi, and Synechococcus showed no changes in activity after 24 h, whereas activity declined by less than 50% in the other species. Incubation of cell extracts for 1 h at 25°C in pH 7.5 buffer did not alter patterns of cell proteins, suggesting that endogenous proteases did not effectively degrade endogenous proteins. Casein zymograms were used to identify >200-and <20-kDa proteases in homogenates of log-phase T. weissflogii; only the smaller protease was found in D. tertiolecta. Antibodies to the ATPase subunit (C) of the conserved, chloroplastic Clp protease from Pisum cross-reacted with proteins in Synechococcus, D. tertiolecta, and I. galbana, but no cross-reactions were found for any species with antibodies against the ClpP subunit from either E. coli or Nicotiana. Our results show that phytoplankton contain a diverse complement of proteases with novel characteristics.     

The function of lymphocyte proteases. Inhibition and restoration of granule-mediated lysis with isocoumarin serine protease inhibitors.

To kill other cells, lymphocytes can exocytose granules that contain serine proteases and pore-forming proteins (perforins). We report that mechanism-based isocoumarin inhibitors inhibited the proteases and inactivated lysis. When inhibited proteases were restored, lysis was also restored, indicating that the proteases were essential for lysis. We found three new lymphocyte protease activities, "Asp-ase,"Met-ase," and "Ser-ase," which in addition to ly-tryptase and ly-chymase, comprise five different protease activities in rat RNK-16 granules. The general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inhibited all five protease activities. Essentially all protease molecules were inactivated by DCI before lysis was reduced, as determined from DCI's second order inhibition rate constants for the proteases, the DCI concentrations, and the times of pretreatment needed to block lysis. The pH favoring DCI inhibition of lysis was the pH optimum for protease activity. Isocoumarin reagents acylate, and may sometimes secondarily alkylate, serine protease active sites. Granule proteases, inhibited by DCI acylation, were deacylated with hydroxylamine, restoring both the protease and lytic activities. Hydroxylamine does not restore alkylated proteases and did not restore the lytic activities after inhibition with 4-chloro-7-guanidino-3-(2-phenylethoxy)-isocoumarin, a more alkylating mechanism-based inhibitor designed to react with tryptases. It is improbable that isocoumarin reagents directly inactivated pore-forming proteins because 1) these reagents require protease activation, 2) their nonspecific effects are alkylating, and 3) alkylated proteins are not restored by hydroxylamine. We conclude that serine proteases participate in lysis when lysis is mediated by the complete assembly of granule proteins.     

Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638.

An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C. At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.     

辐射过程中耐辐射奇球菌蛋白酶变化的检测与分析

采用明胶和酪蛋白底物酶谱法以及荧光酪蛋白底物对紫外线以及γ射线辐射后恢复期耐辐射奇球菌R1(Deinococcus radiodurans R1,DRR1)的蛋白酶变化进行了检测。结果发现,DRR1存在高活性大分子量组成性表达蛋白酶,与Karlin等［16］提出的DRR1蛋白酶为预测高表达蛋白(PHX)的设想一致。DRR1包含大量分子量大于140kD 的明胶降解酶和分子量大于120kD的酪蛋白降解酶,其中活性最高的174kD明胶酶在经SDS变性处理后仍有较高活性,该蛋白酶在DRR1受紫外线辐射和电离辐射后恢复期的表达模式存在差异,在γ射线电离辐射过程中以及电离辐射后恢复的晚期活性较高。此外,还发现一些蛋白酶特异性由辐射所诱导,表明这些蛋白酶可能参与细胞信号通路中蛋白的顺序降解,也提示DRR1损伤修复过程中细胞内存在一个精确的蛋白酶系统。这些蛋白酶的表达与细胞的营养状态相关。同时对一株由本实验室从北京地区土壤中分离到的杆状耐辐射菌RR533.2的明胶和酪蛋白蛋白酶谱进行了测定,结果发现其蛋白酶谱与DRR1相类似。     

Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)

Abstract  The protein digestive capability of the larvae of the longhorn beetle ( Oemona hirta , Coleoptera: Cerambycidae, Fabricius, 1775) was investigated. This species feeds only on wood where there is a high proportion of vascular tissue. The pH of the midgut, the major digestive organ, was alkaline and protein hydrolysis was maximal at alkaline pH. Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases, trypsin and chymotrypsin-like activity, and the exopeptidase, leucine aminopeptidase and the pH curves corresponded to that with protein substrate. Studies using a range of serine protease inhibitors as well as specific inhibitors of metalloproteases, cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids. Control of these insect pests using protease inhibitors is discussed.     

Field bean protease inhibitor preparations,unlike methotrexate,can completely suppress Yoshida sarcoma tumor in rats

Protease inhibitor preparations (PIP) with antitryptic and antichymotryptic activities, isolated from field bean legume as well as doxorubicin and cyclophosphamide could effectively suppress the growth of Yoshida sarcoma ascites tumor cells transplanted in adult rats and prevent their death. As against this, methotrexate and heat-inactivated PIP were ineffective in such rats at varied doses of treatment tried. The percent survival of animals appeared to be related to the purity, treatment mode and the dose of PIP used. Zymographic analysis of the trypsin activated sarcoma cell homagenate revealed the presence of six protease bands in the molecular weight range of 51kD to 206kD. Prolonged interactions of such zymograms with protease inhibitors such as 20mM EDTA or 5mM diisopropyl fluorophosphate (DIFP) or 400 · μg/ml of PIP in reaction buffer indicated that these are not metalloproteases but serine proteases whose activities are inhibited by PIP and DIFP. Since proteases are involved in cell growth regulation and cell transformation, we hypothesize a positive relationship between the field bean protease inhibitor;s blocking action on tumor cell proteases and its tumor suppressing activity     

Comparison of serum protein inhibitors from various mammals, chicken and silkworms against four proteases

1. Protein serum inhibitors against four proteases were compared using eight mammals, chicken and silkworms. 2. The similarity of inhibition spectra and electrophoretograms was found in related mammals. 3. In the silkworm and chicken, inhibitory activities against fungal protease and subtilisin were extremely high. 4. Electrophoretic patterns of inhibitors for chymotrypsin and trypsin were very similar in mammals, but different in the silkworm, that is, mammalian inhibitors seemed to show broader protease specificity. 5. Electrophoretic bands of the silkworm inhibitors showed more dispersed molecular species than those of other animals. 6. Column chromatographic patterns of silkworm inhibitors against four proteases showed more diverse and distinct profiles than those of other animals.     

Preparation and properties of proteases immobilized on anion exchange resin with glutaraldehyde

High activity alkaline protease was obtained when the enzyme was immobilized on Dowex MWA-1 (mesh 20–50) with 10% glutaraldehyde in chilled phosphate buffer (M/15, pH 6.5). Activity yields of the protease and rennet were 27 and 29, respectively. The highest activities appeared at 60°C, pH 10 for alkaline protease and 50°C, pH 4.0 for rennet. The properties of both proteases were not essentially changed by the immobilization except that the K_m values of both enzymes were increased about tenfold as a result of immobilization. Both proteases in the immobilized state were more stable than those in the free state at 60°C. Other peptide hydrolases, β-galactosidase, invertase, and glucoamylase, were successfully immobilized with high activities, but lipase, hexokinase, glucose-6-phosphate dehydrogenase, and xanthine oxidase became inactive.     

根虫瘟霉转寄主过程中毒力相关胞外蛋白酶系的诱导表达

根虫瘟霉Zoophthoraradicans是寄主范围较广的专性昆虫病原真菌。在该菌胞外蛋白酶系与毒力变化关系的研究中,不同寄主来源的根虫瘟霉菌株产胞外蛋白酶水平与其对小菜蛾(Plutellaxylostella)的毒力间无明显的相关性。但转寄主各代菌株随毒力逐步上升产胞外蛋白酶水平也有所增加,两者间有一定的相关性。经活性电泳检测显示,ARSEF1342原始菌株(R0)有分子量分别为148kD,153kD和162kD的三条蛋白酶条带,但转寄主传代菌株(R1~R4)的148kD蛋白酶条带突然消失,而153kD蛋白酶条带则随转寄主传代数增加逐步趋于明显,相关性分析发现153kD和148kD与毒力间具有较好的相关性。这表明根虫瘟霉菌株在转寄主过程中逐渐增加了对新寄主具有较高基质特异性的胞外蛋白酶的诱导表达,从而使转寄主菌株更适应对新寄主的入侵及致病。     

Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization,cDNA cloning,and expressed sequence tag analysis

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.     

A survey of proteases in edible mushrooms with synthetic peptides as substrates

The protease activities in six edible mushrooms were surveyed using synthetic fluorogenic substrates that have different specificities for each protease group. The activity was determined by measuring the fluorogenic intensity of the 7-amino-4-methylcoumarin (AMC) liberated by an enzyme. Various types of activities were found in all mushrooms, and their activities depended largely on the mushroom species, but also on the pH and localization. Flammulina velutipes and Pleurotus eryngii had the widest and highest proteolytic activities among the six mushrooms examined. The proteasome-like protease activities were generally much higher than those of other proteases. High caspase activities, which occur during apoptosis in cells, were detected in two mushrooms, F. velutipes and Hypsizigus marmoreus. The pH optima of the proteolytic activities were largely divided into two groups, acidic pH 5–6 for caspases and neutral to alkaline (pH 6.5–11) for the others. In F. velutipes, higher proteolytic activity was observed in the basement of the stem than in the cap and stem. Purification and characterization of protease were also carried out to identify a protease from Grifola frondosa using t-butyloxycarbonyl-Leu-Arg-Arg-4-methylcoumaryl-7-amide (Boc-LRR-MCA) as the substrate.     

Preliminary characterisation of digestive proteases of the green mirid, Creontiades dilutus (Hemiptera: Miridae)

Protease activities in the secreted saliva, salivary glands and midgut of the green mirid, Creontiades dilutus, were investigated. The saliva and salivary glands had more protease activity than the midgut, but no differences in protease activity levels were detected between male and female mirids, adult mirids and third instar nymphs, or between fed and starved mirids.In the salivary glands, chymotrypsin-like serine proteases predominated, as characterised by inhibitor specificity, basic pH optima, and hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide and N-succinyl-ala-ala-pro-leu p-nitroanilide. The pH optimum of midgut extracts was acidic (pH 4), implying that acidic proteases predominate. However, protease activity was inhibited substantially by both aprotinin and E-64, suggesting the presence of both serine and cysteine proteases in the midgut of the green mirid.     

凡纳滨对虾虾头内源性蛋白酶同源性分析

以凡纳滨对虾 (Litopenaeus vannamei shrimp) 虾头为原料,采用Q- Sepharose F F和Sephadex G-150对虾头内源碱性蛋白酶进行了纯化,通过SDS-PAGE测定分子量为79.95 kD|采用DEAE-Sepharose F.F和Sephadex G-100对内源酸性蛋白酶进行了纯化,通过SDS-PAGE测定分子量为27.45 kD. 利用HPLC-ESI-MS/MS对虾头内源碱性和酸性蛋白酶同源性进行了初步分析,将检测到内源性蛋白酶的部分氨基酸序列分别与不同物种的胰蛋白酶和胃蛋白酶氨基酸序列于Vector NTI suite 8.0软件上进行序列比对. 结果表明,内源性碱性蛋白酶与猪胰蛋白酶具有很高的同源性,均含有氨基酸序列LSSPATLNSRVATVSLPR|内源性酸性蛋白酶与非洲蟾蜍胃亚蛋白酶具有很高的同源性,均含有氨基酸序列EFGLSETEPGTNF.     

Properties of extremely thermostable proteases from anaerobic hyperthermophilic bacteria

Summary Hyperthermostable proteases were characterized from five archaeobacterial species (Thermococcus celer, T. stetteri, Thermococcus strain AN 1, T. litoralis, Staphylothermus marinus) and the hyperthermophilic eubacterium Thermobacteroides proteolyticus. These proteases, which were found to be of the serine type, exhibited a preference for phenylalanine in the carboxylic side of the peptide. The enzymes from Thermococcus stetteri and T. litoralis hydrolysed most substrates (peptides) tested. All proteases were extremely thermostable and demonstrated optimal activities between 80 and 95°C. The pH optimum was either neutral (T. celer, Thermococcus strain AN 1) or alkaline. The protease of Thermobacteroides proteolyticus was optimally active at pH 9.5. Zymogram staining showed the presence of multiple protease bands for all strains investigated.Offprint requests to: G. Antranikian     

Electrophoretic analysis of proteases in Echinostoma Caproni and Echinostoma trivolvis

Gelatin substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze proteases in 14 day-old adults of Echinostoma caproni and Echinostoma trivolvis. At pH 8.0, E. caproni adults showed 2 protease bands at 36 kDa and 58 kDa, whereas E. trivolvis adults showed 6 bands at 39, 64, 77, 96, 120, and 168 kDa. Each species also showed distinct protease banding patterns in their excretory/secretory (E/S) products. The E. caproni E/S proteases were at 36 and 58 kDa, whereas those of E. trivolvis were at 120 and 168 kDa. Further characterization of E. caproni adult proteases revealed 2 bands (58 and 66 kDa) with optimal activity at pH 3.0-4.5 and 3 bands (38, 61, and 96 kDa) that were most active at pH 7.0-8.0. Four low molecular weight bands (19, 21, 25, and 30 kDa) appeared when E. caproni worm extracts were incubated in the presence of CaCl2 at pH 8.0 but were inhibited with ethylenediaminetetraacetic acid and 1,10-phenanthroline. Echinostoma caproni protease bands at 58 and 38 kDa in the whole worm samples and the E/S products and the 36-kDa band in the whole worm samples were inhibited with phenylmethylsulfonyl fluoride. By showing protease differences in addition to recent work on nucleotide differences, this study helps distinguish these 2 related allopatric species of 37-collar-spined Echinostoma.