一种凋亡相关蚯蚓丝氨酸蛋白酶的纯化、活性鉴定及部分性质研究

通过多级柱层析,从赤子爱胜蚓抽提物（一组抗肿瘤活性蛋白成分）中纯化得到凋亡相关丝氨酸蛋白酶1（apoptosis-related serine protease 1, ARSP1）,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)测得其表观分子质量为28 ku.ARSP1非变性PAGE图谱为相连的多条带,质谱图为多头峰,MALDI-TOF-MS测得各主峰相对分子质量为24 645,25 052和25 281,等电聚焦电泳测得等电点pI＜3.8.测得ARSP1 N端25个氨基酸序列为：I(V)IGGT(S)N(D)ASPGEFPWQLSQTRGGSHS,N端序列比较结果显示其与丝氨酸蛋白酶类高度同源.体外实验中,不仅通过凋亡细胞的相差显微观察验证了ARSP1的细胞杀伤活性,而且进一步通过荧光抗体技术对其直接杀伤细胞活性进行了定位研究.Schiff's试剂糖蛋白染色法鉴定ARSP1为糖蛋白（或糖肽）,纤维蛋白平板法测得ARSP1同时具有纤溶酶和纤溶酶原激活酶活性,进一步通过苯甲磺酰氟(PMSF)对其纤溶酶活性的抑制实验,证明属于丝氨酸蛋白酶类.     

Extracellular protease of Natrialba magadii: purification and biochemical characterization

A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60°C in the presence of 1.5 M NaCl. The enzyme was stable and had a broad pH profile (6–12) with an optimum pH of 8–10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated. Received: October 1, 1999 / Accepted: February 1, 2000     

Characterization of a neutral serine protease and its full-length cDNA from the nematode-trapping fungus Arthrobotrys oligospora

A neutral serine protease (designated Aoz1) was purified to homogeneity from a strain of Arthrobotrys oligospora, obtained from soil in Yunnan Province. The purified protein showed a molecular mass of approximately 38?000 Dalton, pI 4.9 and displayed optimal activity at 45 C and pH 6-8. The protein could hydrolyze gelatin, casein and the chromogenic substrate azocoll, and it could immobilize nematodes in vitro (Panagrellus redivivus L. [Goodey]). The level of activity in culture medium was found to increase with increasing gelatin concentration. Scanning electron micrographs demonstrated dramatic structural changes in nematode cuticle treated with the purified protease. A partial peptide sequence obtained by N-terminal sequence analysis was used to design degenerate primers for the isolation of a cDNA gene encoding the mature protease. Analysis of the cDNA and corresponding genomic sequence revealed 97% identity with PII, a gene previously described from A. oligospora, and we conclude that this gene is likely a PII ortholog.     

苜蓿夜蛾丝氨酸蛋白酶基因cDNA序列的克隆与原核表达研究

【目的】丝氨酸蛋白酶(Serine protease,SP)是以丝氨酸为活性中心的重要的蛋白水解酶。在昆虫中,丝氨酸蛋白酶参与消化、发育、先天免疫反应和组织重建等重要的生理过程。本试验以苜蓿夜蛾Heliothis viriplaca为材料,克隆其丝氨酸蛋白酶基因的cDNA序列,再对该基因进行原核表达并对表达产物进行活性测定研究。【方法】从苜蓿夜蛾中肠中提取总RNA,通过RT-PCR和RACE技术,扩增获得丝氨酸蛋白酶基因cDNA全长序列,用大肠杆菌E.coli表达系统进行表达;再对表达的重组蛋白进行变性、纯化与复性,并以BTEE为底物进行活性测定。【结果】克隆得到的苜蓿夜蛾中肠丝氨酸蛋白酶基因命名为Hv SP,该基因已登录Gen Bank,登录号为KT907053。该基因全长1 017 bp,开放阅读框为886 bp,编码295个氨基酸,分子量约为30.8 ku,等电点为8.27,推导的氨基酸序列与其他昆虫丝氨酸蛋白酶氨基酸序列相似性在46%~92%之间。在Tris-HCl缓冲液中,p H为8.5时,复性的重组蛋白活性最高,为28.7 U/m L。荧光定量PCR结果表明,Hv SP基因的m RNA在苜蓿夜蛾的多个组织中特异性表达,且在中肠中表达量最高,但在唾腺中未检测到Hv SP的m RNA表达。【结论】该研究克隆了一个新的苜蓿夜蛾丝氨酸蛋白酶基因的cDNA序列,且原核表达后的重组蛋白经过变性、纯化及复性后具有活性,为进一步探索丝氨酸蛋白酶在昆虫体内的生理生化功能奠定了基础。     

蛋白酶抑制剂对绿豆象幼虫中肠蛋白酶活性的影响

为明确蛋白酶抑制剂对绿豆象幼虫中肠蛋白酶活性的影响,采用室内人工接虫和生化测定的方法,研究了在离体条件和饲喂条件下4种蛋白酶抑制剂对绿豆象幼虫中肠蛋白酶的抑制作用,并测定了绿豆象幼虫取食不同含量的绿豆胰蛋白酶抑制剂(MBTI)的人工绿豆后,其中肠内总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性的变化.结果表明:在离体条件下,供试4种蛋白酶抑制剂对绿豆象幼虫总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶活性均有明显的抑制作用,且浓度越大,抑制效果越显著,其中以20μg·mL^-1的MBTI对3种酶活性的抑制效果最强,3种酶活性分别比对照降低了62.5%、41.2%和38.7%,而卵粘蛋白抑制剂(OI)抑制效果最弱.绿豆象幼虫取食含不同抑制剂的人工绿豆后,中肠内3种酶活性也均受到一定的抑制作用,取食后随龄期的延长,3种酶活性有所升高但仍显著低于对照,且以MBTI的抑制作用最强.当绿豆象幼虫取食不同含量MBTI的人工绿豆后,随MBTI含量的增加,对总蛋白酶活性和类胰蛋白酶活性的抑制作用均逐渐增强,但对类胰凝乳蛋白酶活性的抑制作用并不显著,只有当MBTI含量达20%时,对类胰凝乳蛋白酶活性才表现出明显的抑制作用.     

丙型肝炎病毒丝氨酸蛋白酶特异性结合肽的筛选和鉴定

丙型肝炎病毒丝氨酸蛋白酶在病毒复制和包装中的重要作用使其成为特异性抗病毒药物研究的首选靶标。根据丝氨酸蛋白酶晶体结构特点,用柔性连接子连接NS3丝氨酸蛋白酶结构域和NS4A的核心序列,构建成单链丝氨酸蛋白酶基因并且在大肠杆菌中获得高水平的可溶性表达,纯化后的目的蛋白能够切割重组蛋白底物NS5ab。随后,以单链丝氨酸蛋白酶为靶分子对噬菌体展示的随机十二肽库进行了三轮淘筛,挑选的44个克隆中有37个克隆能够特异性地结合丝氨酸蛋白酶,并且这种结合作用为竞争性ELISA试验结果所支持。对13个克隆进行序列测定,得到6种序列,它们在氨基酸组成上存在明显偏性,富含组氨酸和色氨酸,缺乏酸性氨基酸;6种序列存在一个共有序列。     

HCV丝氨酸蛋白酶小分子底物构建及GST融合表达

丝氨酸蛋白酶是丙型肝炎病毒重要的功能蛋白和药物作用靶点,其通过分子内（cis）和分子间（trans）方式催化水解前体蛋白,释放病毒功能蛋白。目的：为深入研究病毒蛋白酶活性和抑制剂鉴定需要,实验研究参照丙型肝炎病毒1a亚型菌株蛋白酶天然底物的氨基酸序列特点,设计了一段包含两个天然底物酶切位点的小分子多肽2S,并进行了原核表达。方法：利用PCR方法,合成2S小分子多肽基因,目的基因两端引入BamH I和EcoR I两个限制性酶切位点,双酶切后将基因与表达载体pGEX-4T-2重组,转化大肠杆菌DH5α,经化学诱导进行GST融合蛋白表达,通过亲和层析柱纯化目的蛋白。纯化的GST 2S融合蛋白在体外反应系统进行酶切鉴定,SDS-PAGE和ELISA鉴定酶切结果。结果：PCR合成的小分子底物多肽2S基因,经与表达载体重组后测序,证实基因序列正确。采用0.5mmol/L浓度的IPTG诱导工程菌过夜,获得表达的目的蛋白,经分离纯化得到融合蛋白GST-2S。GST-2S在体外磷酸盐缓冲系统中与丝氨酸蛋白酶反应,15%SDS-PAGE鉴定酶切产物,证实融合蛋白底物条带明显消失,ELISA结果同样说明融合蛋白的底物活性。结论：含有两个天然底物酶切位点的小分子多肽可以替代病毒天然底物,实验结果为丙型肝炎病毒丝氨酸蛋白酶活性研究和酶抑制剂研究奠定了方法学基础。     

An apoptosis-inducing serine protease secreted by the entomopathogenic nematode Steinernema carpocapsae

Steinernema carpocapsae is an insect parasitic nematode able to parasitise and kill the host within 48 h. Secreted products (ESP) of the parasitic stage of a virulent strain contain higher amounts of proteolytic activity than a low virulence strain, suggesting proteases are involved in virulence. From the ESP we purified a protein (Sc-SP-3) with a M_r of 30 kDa and a pI of 7 that cleaved the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA and was inhibited by phenylmethanesulfonyl fluoride, benzamidine and chymostatin, thus indicating that it belongs to the chymotrypsin-like serine protease family. Sc-SP-3 has a V_max of 0.3 mM min⁻¹ ml⁻¹ and K_m of 6.6 × 10⁻⁴ M, with maximum activity at pH 8 and 40 °C. The full-length cDNA was obtained using degenerate oligonucleotides for serine proteases. This open reading frame encodes a preproprotein containing a putative signal peptide composed of 16 amino acid residues, a prodomain of 40 residues and a mature protease domain of 261 residues, including the catalytic triad His/Asp/Ser characteristic of trypsin-like serine proteases. The N-terminal sequence and the peptide masses fingerprint obtained by MALDI-TOF–MS for the purified protein matched the cDNA. Gene expression analysis by quantitative real-time-PCR showed that this gene is expressed only during the parasitic stage and that pre-invasive nematodes inside the mid-gut expressed higher amounts of Sc-SP-3 than those that already enter the haemocoel. Sc-SP-3 caused histolysis in the insect mid-gut. In vitro assays demonstrated that Sc-SP-3 digested extracellular proteins and induced apoptosis in Sf9 insect cells, thus suggesting Sc-SP-3 is a multifunctional chymotrypsin-like protease involved in pathogenesis.     

Inhibition of the protease activity of influenza virus RNA polymerase PA subunit by viral matrix protein

Influenza virus PA is a subunit of RNA-dependent RNA polymerase. We demonstrated that PA has a unique chymotrypsin-like serine protease activity with Ser624 as an active site. To obtain further insight into the role of the protease activity of PA in viral proliferation, we examined the interaction between PA and matrix protein (M1). Both M1 purified from virion and hexa-histidine-tagged M1 expressed in Escherichia coli bound to PA. Hexa-histidine-tagged M1 pulled down PA. The interaction of PA with M1 was sensitive to ionic strength, suggesting that the interaction is formed by electrostatic force. Using Suc-Leu-Leu-Val-Tyr-MCA, a specific substrate for PA protease, M1 was demonstrated to inhibit the amidolytic activity of PA, whereas M1 did not inhibit that of chymotrypsin or trypsin at all. These results suggest that M1 binds to and inhibits the amidolytic activity of PA.     

Epi p 1, an allergenic glycoprotein of Epicoccum purpurascens is a serine protease

Epicoccum purpurascens (EP) is a ubiquitous saprophytic mould, the inhalant spores and mycelia of which are responsible for respiratory allergic disorders in 5-7% of population worldwide. The diagnosis/therapy of these disorders caused by fungi involves the use of standardized and purified fungal extracts. A 33.5 kDa glycoprotein, Epi p 1 released histamine from whole blood cells of EP allergic patients at a concentration of 50-ng protein. The high specific IgE values detected in EP hypersensitive sera indicated that Epi p 1 is capable of mediating type I hypersensitive reaction in predisposed individuals. It also showed protease activity by virtue of its dose dependent cleavage of serine protease specific synthetic substrate, N-benzoyl arginine ethyl ester hydrochloride (BAEE). The serine protease nature of Epi p 1 was confirmed by its N-terminal sequence (ADG/FIVAVELD/STY) homology to a subtilisin like serine protease. The protease activity of Epi p 1 may be responsible for making its way into the system of pre-disposed individuals through epithelial cell detachment and the histamine releasing ability by cross-linking of IgE antibodies on cell surface is the cause of its allergenic nature.     

Purification and partial characterization of chymotrypsin-like proteases from the digestive gland of the scallop Pecten maximus

Three variants of a chymotrypsin-like protease were purified from scallop digestive glands successively by ion-exchange, gel filtration and high-performance liquid chromatographies. Enzyme activity was detected using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a specific synthetic substrate for chymotrypsin. This proteinase was inhibited by chymostatin, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. Estimated molecular mass of the purified enzyme is around 32 kDa. These isoenzymes exhibit very low activities in hydrolyzing small synthetic specific substrates used for trypsic, elastolytic and collagenolytic measurements and referred mainly to a chymotrypsin-like proteinase. Very few differences were measured concerning pH profiles among the three isoenzymes. Stability is higher at low temperature for two variants. An N-terminal analysis was performed on one variant (B) among the three isoenzymes. The alignment of the N-terminal amino acid sequence indicates some homologies with abalone chymotrypsin-like protein and arthropod chymotrypsin proteases as well as with vertebrate serine protease counterparts (trypsin, chymotrypsin and elastase).     

Purification, biochemical and molecular analysis of a chymotrypsin protease with prophenoloxidase suppression activity from the entomopathogenic nematode Steinernema carpocapsae

A chymotrypsin serine protease (designated Sc-CHYM) was purified by gel filtration and anion-exchange chromatography from excretory-secretory products of parasitic stage Steinernemacarpocapsae. The purified protease had an apparent molecular mass of 30 kDa and displayed a pI of 5.9. This protease demonstrated high activity against the chymotrypsin-specific substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and was highly sensitive to the inhibitor aprotinin. This protease digested the chromogenic substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide with K_m, V_max and k_cat values of 409 μM/min, 0.389 μM/min and 24.9 s⁻¹, respectively. The protease was most active at pH 8.0 and 35 °C, and its proteolytic activity was almost completely reduced after incubation at 75 °C for 30 min. In vitro, this enzyme suppressed prophenoloxidase activity. In vivo, demonstration of encapsulation and melanization by purified chymotrypsin imbibed beads showed it could prevent hemocyte encapsulation and melanization by 12 and 24 h, respectively. Sequence comparison and evolutionary marker analysis showed that the putative protein was a chymotrypsin-like protease with potential degradative, developmental and fibrinolytic functions. Expression pattern analysis revealed that the gene expression of Sc-CHYM was up-regulated in the parasitic stage. Sc-CHYM was clustered with several insect chymotrypsins and formed an ancestral branch in the phylogenetic tree, suggesting that Sc-CHYM branched off at an early stage of cluster divergence. The results of this study suggest that Sc-CHYM is a new member of the chymotrypsin serine protease family and that it might act as a virulence factor in host-parasite interactions.     

一种新的丙型肝炎病毒单链丝氨酸蛋白酶基因的构建、表达与鉴定

根据丙型肝炎病毒 (HCV)丝氨酸蛋白酶晶体结构特点 ,设计并构建了一种新的单链型丝氨酸蛋白酶分子 .该分子由辅因子NS4A的核心序列、柔性连接子GSGS和NS3丝氨酸蛋白酶结构域组成 .利用设计的 3条引物 ,通过 2轮PCR获得单链丝氨酸蛋白酶基因 ,插入原核表达载体pQE30中 ,转化大肠杆菌M15 ,获得重组克隆 .经低剂量诱导和低温培养 ,目的基因获得高水平可溶表达 .以金属螯合层析法纯化的重组蛋白纯度达 95 %以上 .间接ELISA法检测 98份血清证实 ,该蛋白具有良好的抗原性和特异性 ;以重组蛋白底物NS5ab和单链丝氨酸蛋白酶建立了简便、实用的丝氨酸蛋白酶体外活性检测系统 ;以该系统观察了PMSF和EDTA对蛋白酶活性的影响 .结果表明 ,PMSF能够抑制蛋白酶的酶切活性 ,而EDTA不能抑制酶的活性 .单链型HCV丝氨酸蛋白酶的成功表达以及体外活性检测系统的建立 ,为丝氨酸蛋白酶抑制剂的研制奠定了物质基础 .     

Isolation and characterization of Perkinsus marinus proteases using bacitracin–sepharose affinity chromatography

Extracellular proteases were isolated from the cell-free culture supernatant of the oyster-pathogenic protozoan, Perkinsus marinus, by bacitracin–sepharose affinity chromatography. The purified protease fractions contained >75% of the protease activity initially loaded onto the column with very high specific activity that corresponded to 8–11-fold level of protease enrichment. The isolated proteases hydrolysed a variety of protein substrates including oyster plasma. All of the isolated P. marinus proteases belonged to the serine class of proteases. Inhibitor studies involving spectrophotometric assay and gelatin gel electrophoresis showed high levels of inhibition in the presence of the serine protease inhibitors PMSF, benzamidine and chymostatin, whereas inhibitors of cysteine, aspartic, and metalloproteases showed little or no inhibition. Spectrophotometric assays involving serine-specific peptide substrates further revealed that the isolated proteases belong to the class of chymotrypsin-like serine proteases. A 41.7 kDa monomeric, N-glycosylated, serine protease (designated Perkinsin) has been identified as the major P. marinus extracellular protease.     

Purification and characterization of extracellular alkaline serine protease from Stenotrophomonas maltophilia strain S-1

AIMS: The present study was conducted by screening soil bacteria in an attempt to isolate a bacterium that produced extracellular alkaline protease, and for purification and characterization of the protease. METHODS AND RESULTS: Soil bacteria were screened by growth on casein as the sole carbon source. Characterization of a strain isolated from soil of Abashiri, Japan indicated a taxonomic affiliation to Stenotrophomonas maltophilia, and was named S-1 strain. The purified S-1 protease, designed S. maltophilia Protease-1 (SmP-1), exhibited an optimal pH of 12.0, optimal reaction temperature of 50 degrees C and a molecular mass of approximately 40 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cleavage sites of the oxidized-insulin B chain by SmP-1 were identified as Leu6-Cys7, Cys7-Gly8, Tyr16-Leu17 and Leu17-Val18. The N-terminal amino acid sequence of the purified alkaline protease was determined as NH2-SASAPMVSGVAALVLE. CONCLUSION: A novel extracellular alkaline serine protease was isolated from S. maltophilia strain S-1. The optimal pH of the proteolytic activity was pH 12.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The extremely high optimal pH and heat stability of the alkaline serine protease SmP-1 might make it widely applicable to food and other industries.     

Alkaline protease activity associated with iridescent virus type 6

Abstract An alkaline protease activity has been found to be associated with Iridovirus type 6. The enzyme showed a pH optimum of 10–10.5 with azocoll and hemoglobin but was essentially inactive on casein. From inhibitor studies the enzyme behaved like a serine protease. An M _r value of about 11500 was determined by SDS-PAGE.     

The two main endoproteases present in dark-induced senescent wheat leaves are distinct subtilisin-like proteases

We have previously reported the occurrence of two serine endoproteases (referred to as P1 and P2) in dark-induced senescent wheat (Triticum aestivum L.) leaves. P1 enzyme was already purified and identified as a subtilisin-like serine endoprotease (Roberts et al. in Physiol Plant 118:483–490, 2003). In this paper, we demonstrate by Western blot analysis of extracts obtained from dark-induced senescent leaves that an antiserum raised against P1 was able to recognise a second protein band of 78 kDa which corresponded to P2 activity. This result suggested that both enzymes must be structurally related. Therefore, we purified and characterised P2 activity. According to its biochemical and physical properties (inhibition by chymostatin and PMSF, broad pH range of activity, thermostability and ability to hydrolyse Suc-AAPF-pNA) P2 was classified as a serine protease with chymotrypsin-like activity. In addition, P2 was identified by mass spectrometry as a subtilisin-like protease distinct from P1. Western blot analysis demonstrated that P1 appeared in extracts from non-detached dark-induced senescent leaves but was undetectable in leaves senescing after nitrogen (N) deprivation. In contrast, P2 was already present in non-senescent leaves and showed increased levels in leaves senescing after N starvation or incubation in darkness. P1 signal was detected at late stages of ethephon or methyl jasmonate-induced senescence but was undetectable in senescent leaves from plants treated with abscisic acid. None of the three hormones have any effect on P2 protein levels. These results indicate that despite their biochemical and structural similarities, both enzymes are probably involved in different physiological roles.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Physicochemical and biological properties of an extracellular serine protease of Aeromonas sobria

Previously, we cloned a protease gene of Aeromonas sobria, determined its nucleotide sequence and established a method of purifying its product. In this study, we examined the properties of the purified protease. The protease was temperature-labile and had an optimal pH of 7.5. Metallo-protease inhibitors and a cysteine protease inhibitor did not block the proteolytic activity of the enzyme. The treatment with reagents to modify sulfhydryl group did not reduce the activity. But, serine protease inhibitors did, showing that it was a serine protease. Subsequently, we examined the ability of the protease to enhance vascular permeability in dorsal skin. The protease showed activity and the reaction was inhibited by a simultaneously injected antihistaminic agent. Histopathological examination showed that mast cells appeared around the site where the protease was injected. These findings show that the vascular permeability-enhancing effect of the protease is due to histamine released at the site. Furthermore, we found that a soybean trypsin inhibitor (Kunitz) did not block the proteolytic action of the protease in vitro, but inhibited its vascular permeability-enhancing activity in skin. This suggests that a trypsin-like protease from skin mediates the activity of the protease to enhance its vascular permeability.     

A novel serine protease complexed with α2-macroglobulin from skeletal muscle of lizard fish (Saurida undosquamis)

A novel fish muscle serine protease named muscle soluble serine protease (MSSP) was purified from the soluble fraction of lizard fish (Saurida undosquamis: Synodontidae) muscle by ammonium sulfate fractionation followed by four steps of column chromatographies. In native-PAGE, the purified enzyme appeared as a single band with an estimated mol. mass of approximately 380 kDa by gel filtration. In SDS-PAGE under reducing conditions, the purified enzyme migrated as two protein bands at 110 and 100 kDa, named subunits A and B, respectively. The 20 residues of N-terminal amino acid sequence of subunit B showed 70% of homology to β-chain of carp α₂-macroglobulin-1. Moreover, both subunits A and B showed immunoreactivity with anti carp α₂-macroglobulin antibody. Purified MSSP was inactivated by Pefabloc SC, aprotinin, benzamidine and TLCK, but not by α₁-antitrypsin. After acid treatment (pH 2, 24 h), however, the enzyme activity eluted at 14 kDa from Sephacryl S-200 carried out under acidic conditions was inhibited by α₁-antitrypsin. Lizard fish MSSP most rapidly hydrolyzed Boc-Val-Pro-Arg-MCA and Boc-Gln-Arg-Arg-MCA, but did not hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA and Suc-Ala-Ala-Pro-Phe-MCA, and was not suppressed either by E-64, pepstatin A and ethylenediaminetetraacetic acid (EDTA). These results indicate that the purified MSSP is a serine protease complexed with α₂-macroglobulin, and the entrapped protease was dissociated by the acid treatment. Purified and free MSSPs were most active at pH 10.0 and 9.0, respectively. Purified MSSP degraded myofibrillar proteins and casein but time courses of degradation of these substrates by the enzyme differed.     

Identification of a Chymotrypsin-Like Mast Cell Protease in Rat Brain Capable of Generating the N-Terminus of the Alzheimer Amyloid β-Protein

Abstract: Cleavage after Met⁵⁹⁶ of the β-amyloid precursor protein to generate the N-terminus of β-protein indicates the activity of a protease having chymotrypsin-like specificity. A chymotrypsin-like protease is further implicated in Alzheimer's disease by the increased synthesis of the protease inhibitor α₁-antichymotrypsin in pathologically affected brain regions and by the presence in the amyloid deposits of inactivated forms of α₁-antichymotrypsin (indicating irreversible binding to a target chymotrypsin-like protease). In the present report, we have purified from rat brain a chymotrypsin-like protease that (a) binds with high affinity to human α₁-antichymotrypsin, (b) proteolytically generates a β-protein-containing C-terminal fragment from full-length recombinant human β-amyloid precursor protein, and (c) selectively cleaves methoxysuccinyl-Glu-Val-Lys-Met-p-nitroanilide (a substrate modeling the protease recognition domain for the β-protein N-terminal cleavage site). Amino acid sequences of tryptic fragments of the purified rat brain chymotrypsin-like protease indicate an identity with rat mast cell protease I. Moreover, the ontogeny and compartmentalization of rat brain chymotrypsin-like protease are consistent with those of connective tissue-type mast cells in the meningeal and intracortical perivasculature. Because these areas in human brain form extensive β-amyloid deposits in Alzheimer's disease, Down's syndrome, and hereditary cerebral hemorrhage with amyloidosis of Dutch origin, the present findings suggest that a brain mast cell chymotrypsin-like protease may participate in generating perivascular β-protein, which ultimately aggregates into β-amyloid deposits.