共查询到20条相似文献,搜索用时 15 毫秒
1.
George KM Schule T Sandoval LE Jennings LL Taylor P Thompson CM 《The Journal of biological chemistry》2003,278(46):45512-45518
Two types of polyclonal antibodies were generated from (a) a decapeptide sequence that includes the active site serine of acetylcholinesterase (anti-AChE10S) and (b) the identical decapeptide sequence phosphorylated at the active site serine of acetylcholinesterase (anti-AChE10SP). The anti-AChE10S antiserum was found to specifically recognize native, control, and vehicle-treated recombinant mouse AChE (rMoAChE) but did not recognize rMoAChE that was phosphorylated by the four organophosphate (OP) compounds tested. Conversely the anti-AChE10SP antiserum recognized phosphoserine rMoAChE that resulted from reaction with phosphorous oxychloride (POCl3) but did not recognize native or vehicle-treated rMoAChE. Anti-AChE10SP also did not recognize OP-AChE conjugates that resulted from the reaction of rMoAChE with other OP compounds that afford neutral or monoanionic phosphoserine groups thereby indicating a high specificity for a precise OP conjugate. Antisera recognition correlated well with the rates of enzyme inhibition, aging, and oxime-induced reactivation indicating these antisera can both quantify the extent and type of inhibition and also differentiate between select mechanisms of inhibition. The ability to discern mechanistic differences between native AChE and OP-AChE conjugates suggests that these antisera can be used to identify biomarkers of OP exposure in a mechanism-based approach. 相似文献
2.
S Chevallier P Goeltz P Thibault D Banville J Gagnon 《The Journal of biological chemistry》1992,267(12):8192-8199
A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families. 相似文献
3.
Isolation and sequence of an active-site peptide containing a catalytic aspartic acid from two Streptococcus sobrinus alpha-glucosyltransferases 总被引:16,自引:0,他引:16
G Mooser S A Hefta R J Paxton J E Shively T D Lee 《The Journal of biological chemistry》1991,266(14):8916-8922
An active-site peptide containing an aspartic acid implicated in catalysis has been isolated and sequenced from two Streptococcus sobrinus extracellular glucosyltransferases: sucrose:1,3-alpha-D-glucan 3-alpha-D-glucosyltransferase (GTase-I) and sucrose:1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase (GTase-S). The sequenced peptides, tagged with radiolabeled glucose, were isolated from a pepsin digest of a stabilized glucosylenzyme complex prepared by rapidly denaturing a reaction of enzyme and radiolabeled sucrose. The glucosyl linkage had previously been characterized as a beta-anomer bound to an active-site carboxyl group. Purified GTase-I and GTase-S glucosyl-peptides had the following similar but not identical sequences: GTase-I, Asp-Ser-Ile-Arg-Val-Asp-Ala-Val-Asp; and GTase-S, Asp-Gly-Val-Arg-Val-Asp-Ala-Val-Asp. Each has 3 aspartic acids as potential sites of glucose conjugation, but the relevant residue was not identified in sequence analysis because the highly base-labile glucosyl bond was cleaved in the first sequence cycle. As an alternative, the GTase-I glucosyl-peptide was partially digested at the N terminus with cathepsin C and at the C terminus with carboxypeptidase P. Analysis of the truncated products by fast atom bombardment mass spectrometry localized the glucosyl group to Asp-6 i the GTase-I peptide. In the native enzyme, this sequence is found near the N terminus, well-removed from the glucan-binding site located on a 60-kDa domain at the C terminus. The catalysis-dependent method of incorporating a glucosyl label implicates the aspartic acid as the residue involved in stabilizing an oxocarbonium ion transition state. The peptide segment is highly conserved and homologous to a peptide from sucrase-isomaltase labeled by site-directed irreversible inhibition and peptide segments common to a broad array of alpha-glucosidases and related transferases. 相似文献
4.
Active-site tryptic peptides were isolated from three genetic types of human serum cholinesterase. The active-site peptide was identified by labeling the active-site serine with [3H] diisopropylfluorophosphate. Peptides were purified by high-performance liquid chromatography. Amino acid composition and sequence analysis showed that the peptide from the usual genotype contained 29 residues with the sequence Ser-Val-Thr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala-Ser-Val-Ser-Leu-His-Leu-Leu-Ser-Pro-Gly-Ser-His-Ser-Leu-Phe-Thr-Arg. The active-site serine was the eighth residue from the N- terminal. The peptide containing the active-site serine from the atypical genotype contained 22 residues with the sequence Ser-Val-Thr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala-Ser-Val-Ser-Leu-His-Leu-Leu-Ser-Pro-Gly. The peptide from the atypical-silent genotype contained eight residues with the sequence Gly-Glu-Ser-Ala-Gly-Ala-Ala-Ser. Thus, the sequences of the atypical and atypical-silent active-site peptides were identical to the corresponding portions of the usual peptide.This work was supported by U.S. Army Medical Research and Development Command Contract DAMD 17-82-C-2271 (to O.L.) and NIH Grant GM 27028 (to B.N.L.). 相似文献
5.
Nisole A Lussier FX Morley KL Shareck F Kazlauskas RJ Dupont C Pelletier JN 《Protein expression and purification》2006,46(2):274-284
Acetyl xylan esterase A (AxeA) from Streptomyces lividans belongs to a large family of industrially relevant polysaccharide esterases. AxeA and its truncated form containing only the catalytically competent domain, AxeA(tr), catalyze both the deacetylation of xylan and the N-deacetylation of chitosan. This broad substrate specificity lends additional interest to their characterization and production. Here, we report three systems for extracellular production of AxeA(tr): secretion from the native host S. lividans with the native signal peptide, extracellular production in Escherichia coli with the native signal peptide, and in E. coli with the OmpA signal peptide. Over five to seven days of a shake flask culture, the native host S. lividans with the native signal peptide secreted AxeA(tr) into the extracellular medium in high yield (388 mg/L) with specific activity of 19 U/mg corresponding to a total of 7000 U/L. Over one day of shake flask culture, E. coli with the native secretion signal peptide produced 84-fold less in the extracellular medium (4.6 mg/L), but the specific activity was higher (100 U/mg) corresponding to a total of 460 U/L. A similar E. coli culture using the OmpA signal peptide, produced 10mg/L with a specific activity of 68 U/mg, corresponding to a total of 680 U/L. In 96-well microtiter plates, extracellular production with E. coli gave approximately 30 and approximately 86 microg/mL in S. lividans. Expression in S. lividans with the native signal peptide is best for high level production, while expression in E. coli using the OmpA secretion signal peptide is best for high-throughput expression and screening of variants in microtiter plate format. 相似文献
6.
Mitsuhiro Nakamura Yuichi Oba Tatsuya Mori Kazuki Sato Yukisato Ishida Tsukasa Matsuda Hideshi Nakamura 《Biochemical and biophysical research communications》2002,290(3):1037-1041
mu-Conotoxin GIIIA, one of the strong peptide toxins in the cone shell, preferentially blocks the skeletal muscle-type sodium channels in vertebrates. The toxicity of mu-conotoxin GIIIA is nearly equal to that of tetrodotoxin. The generation of an antibody for the native toxins is analytically useful, but practically difficult due to its high toxicity to animals. In this study, we generated the polyclonal antibody for mu-conotoxin GIIIA using a specific conjugation method in which the immunogen was detoxified while retaining the active-site structure for the sodium channels. ELISA analysis showed that the generated antibody recognized the native toxin folded with three disulfide bridges, but not the linear one. Furthermore, the physiologically active mutants of GIIIA were recognized while the inactive mutants were not, suggesting that the newly generated antibody can selectively recognize the physiologically active toxins. These methods for generating an antibody against peptide toxins will be applicable to other peptide toxins. 相似文献
7.
Expression of human gelatinase B in Pichia pastoris. 总被引:1,自引:0,他引:1
N Roy S Padmanabhan M Smith L Shi M Navre G Das 《Protein expression and purification》1999,16(2):324-330
Full-length human gelatinase B (FLGelB) and its C-terminal truncated form (dGelB) were expressed in Pichia pastoris strain GS115, using the Saccharomyces cerevisiae Mat alpha signal peptide. In both cases, a high level of the secreted protein could be detected by SDS-PAGE. The truncated gene was also expressed using the human gelatinase B native signal peptide. Secretion using the Mat alpha signal peptide was significantly greater than that from the native signal peptide. The recombinant products were purified and characterized biochemically. The recombinant proteins, FLGelB and dGelB, were found to have similar biochemical properties and activity to that of the human gelatinase B native protein. 相似文献
8.
The active-site and amino-terminal amino acid sequence of bovine intestinal alkaline phosphatase 总被引:2,自引:0,他引:2
The active site of bovine intestinal alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) was labeled with [32P]Pi, a radioactive CNBr peptide was isolated and the amino acid sequence was determined. The sequence of the active-site peptide has limited homology (26%) with the active-site sequence of Escherichia coli alkaline phosphatase except for the ten residues immediately flanking the active-site serine (70%). A possible amino acid sequence deduced from the amino acid composition of an active-site tryptic peptide from human placental alkaline phosphatase is very similar to the bovine intestinal active-site sequence. The amino-terminal sequence of bovine intestinal alkaline phosphatase is homologous (69%) with the human placental enzyme but not with the E. coli phosphatase. 相似文献
9.
R J Raemaekers L de Muro J A Gatehouse A P Fordham-Skelton 《European journal of biochemistry》1999,265(1):394-403
Phytohemagglutinin (Phaseolus vulgaris agglutinin; PHA; E- and L-forms) and snowdrop lectin (Galanthus nivalis agglutinin; GNA) were expressed in Pichia pastoris using native signal peptides, or the Saccharomyces alpha-factor preprosequence, to direct proteins into the secretory pathway. PHA and GNA were present as soluble, functional proteins in culture supernatants when expressed from constructs containing the alpha-factor preprosequence. The recombinant lectins, purified by affinity chromatography, agglutinated rabbit erythrocytes at concentrations similar to the respective native lectins. However, incomplete processing of the signal sequence resulted in PHA-E, PHA-L and GNA with heterogenous N-termini, with the majority of the protein containing N-terminal extensions derived from the alpha-factor prosequence. Polypeptides in which most of the alpha-factor prosequence was present were also glycosylated. Inclusion of Glu-Ala repeats at the C-terminal end of the alpha-factor preprosequence led to efficient processing N-terminal to the Glu-Ala sequence, but inefficient removal of the repeats themselves, resulting in polypeptides with heterogenous N-termini still containing N-terminal extensions. In contrast, PHA expressed with the native signal peptide was secreted, correctly processed, and also fully functional. No expression of GNA from a construct containing the native GNA signal peptide was observed. The PHA-E signal peptide directed correct processing and secretion of both GNA and green fluorescent protein (GFP) when used in expression constructs, and is suggested to have general utility for synthesis of correctly processed proteins in Pichia. 相似文献
10.
Tat (twin arginine translocation) systems transport folded proteins across the thylakoid membrane of chloroplasts and the plasma membrane of most bacteria. Tat precursors are targeted by hydrophobic cleavable signal peptides with twin arginine (RR) motifs. Bacterial precursors possess an extended consensus, (S/T)RRXFLK, of which the two arginines and the phenylalanine are essential for efficient transport. Thylakoid Tat precursors possess twin arginines but lack the consensus phenylalanine. Here, we have characterized two stages of precursor binding to the thylakoid Tat signal peptide receptor, the 700-kDa cpTatC-Hcf106 complex. The OE17 precursor tOE17 binds to the receptor by RR-dependant electrostatic interactions and partially dissociates during blue native gel electrophoresis. In addition, the signal peptide of thylakoid-bound tOE17 is highly exposed to the membrane surface, as judged by accessibility to factor Xa of cleavage sites engineered into signal peptide flanking regions. By contrast, tOE17 containing a consensus phenylalanine in place of Val(-20) (V - 20F) binds the receptor more strongly and is completely stable during blue native gel electrophoresis. Thylakoid bound V - 20F is also completely protected from factor Xa at the identical sites. This suggests that the signal peptide is buried deeply in the cpTatC-Hcf106 binding site. We further provide evidence that the proton gradient, which is required for translocation, induces a tighter interaction between tOE17 and the cpTat machinery, similar to that exhibited by V - 20F. This implies that translocation involves a very intimate association of the signal peptide with the receptor complex binding site. 相似文献
11.
N Koganesawa T Aizawa K Masaki A Matsuura T Nimori H Bando K Kawano K Nitta 《Protein engineering》2001,14(9):705-710
Expression systems of human and silkworm lysozymes were constructed using the methylotrophic yeast Pichia pastoris as a host. The leader sequence and its prepro peptide of alpha-factor (a peptide pheromone derived from yeast) and the native signal sequences of these lysozymes, were used as secretion signals. When the alpha-factor leader is used as the signal sequence, human lysozyme is secreted at a much higher level than is silkworm lysozyme. On the other hand, silkworm lysozyme, when its native signal is used, is secreted more efficiently than human lysozyme. Therefore, we expected that human lysozyme cDNA with a silkworm native signal would be secreted more efficiently than human lysozyme with its native signal. However, its level of expression was not increased. This result indicates that the native signal of silkworm lysozyme does not promote the secretion of the lysozyme, but rather alpha-factor leader inhibits the secretion. Silkworm lysozyme with the alpha-factor leader is so unstable that it could be easily attacked by some proteases and our findings suggest that the level of expression of heterologous protein with signal peptides and its stability are greatly affected by the selection of the appropriate secretion signal sequence. 相似文献
12.
cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue 总被引:8,自引:0,他引:8
Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80,751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. [3H]Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease. 相似文献
13.
M P Caulfield L T Duong R O'Brien J A Majzoub M Rosenblatt 《Molecular endocrinology (Baltimore, Md.)》1988,2(5):452-458
The chemically synthesized signal peptide (native-sequence signal peptide) of preproparathyroid hormone exhibits signal sequence-like activity by inhibiting the translocation/processing of precursor proteins to their mature forms in an in vitro translation system. In order to prepare a biologically functional radiolabeled form of this peptide, we undertook structure-function studies of the native-sequence signal peptide. Since conventional iodination of peptides is performed under oxidizing conditions, chemical design efforts were focused on the oxidation-labile residues, methionine and cysteine, present in the native sequence. Substitution of the three methionines with norleucine and the single cysteine with alanine yielded a surfur-free analog, [Nle-(-25), Nle-(-21),Nle-(-18),Ala-(-14),D-Tyr-(+1)]pre-proPTH-(-29-+1)amide, which is resistant to oxidation and active in the inhibition of processing assay. An interaction between the signal region and one of the components of the intracellular secretory apparatus, signal recognition particle (SRP), was demonstrated: iodinated sulfur-free analog was cross-linked (using the homo-bifunctional reagent disuccinimidyl suberate) to the 54 kilodalton (kDa) subunit of SRP. The 68 kDa and 72 kDa subunits of SRP were also labeled, but to a lesser extent, by the iodinated peptide. 相似文献
14.
目的:构建呈现人白细胞介素-13(interleukin-13,IL-13)抗原肽的Qβ噬菌体病毒样颗粒(virus-like particles,VLPs)疫苗。方法:将人IL-13抗原肽经基因重组插入Qβ噬菌体衣壳蛋白(CP)的C端。在BL21细菌中,经IPTG诱导CP及C端接有IL-13抗原肽的CP(CP/IL-13)同时表达。以硫酸铵沉淀及蔗糖密度梯度离心进行VLPs纯化及分析嵌合VLPs的存在,以HPLC分析VLPs纯度,以电镜观察颗粒形态。小鼠经皮下免疫VLPs后采集血清,以ELISA检测人IL-13特异性Ig G抗体水平。结果:重组蛋白CP与CP/IL-13获得成功表达,两者在密度梯度离心中有一致的、与QβVLPs相同的沉降行为,而CP/IL-13单独无Qβ颗粒行为。经纯化获得了高纯度颗粒,嵌合颗粒与Qβ颗粒形态相似。此外,该VLPs疫苗诱导小鼠产生了IL-13特异的抗体应答。结论:利用共表达策略可成功构建呈现人IL-13抗原表位的嵌合VLPs,为以主动免疫方式调控IL-13在疾病中的病理作用,提供了具有临床应用潜能的疫苗形式。 相似文献
15.
Amino acid sequence of the pyruvate and the glyoxylate active-site lysine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase 总被引:1,自引:0,他引:1
Pure 2-keto-4-hydroxyglutarate aldolase of Escherichia coli, a "lysine-type" trimeric enzyme which has the unique properties of forming an "abortive" Schiff-base intermediate with glyoxylate (the aldehydic product/substrate) and of showing strong beta-decarboxylase activity toward oxalacetate, binds any one of its substrates (2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate) in a competitive manner. To determine whether the substrates bind at the same or different (juxta-positioned) sites and what degree of homology might exist between the active-site lysine peptide of this enzyme and that of other lysine-type (Class I) aldolases or beta-decarboxylases, the azomethine formed separately by this aldolase with either [14C]pyruvate or [14C]glyoxylate was reduced with CNBH3-. After each enzyme adduct was digested with trypsin, the 14C-labeled peptide was isolated, purified, and subjected to amino acid analysis and sequence determination. In each case, the same 14-amino acid lysine-peptide was isolated and found to have the following primary sequence: Glu-Phe-*Lys-Phe-Phe-Pro-Ala-Glu-Ala-Asn-Gly-Gly-Val-Lys (where * = the active-site lysine). Hence, glyoxylate competes for, and inhibits aldolase activity by reacting with, the one active-site lysine residue/subunit. This active-site lysine peptide has a high degree (65%) of homology with that of 2-keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida but is not similar to that of any Class I fructose-1,6-bisphosphate aldolase or of acetoacetate beta-decarboxylase of Clostridium acetobutylicum. Furthermore, it was found that extensive reaction of glyoxylate with the N-terminal amino group of this enzyme may well be general complicating factor in sequence studies with proteins plus glyoxylate. 相似文献
16.
hK-Fc融合蛋白的改良、表达及其生物活性的分析 总被引:1,自引:0,他引:1
为了延长人激肽释放酶(hK)的血清半衰期,提高分泌蛋白的产率,制备了重组激肽释放酶-IgG1 Fc融合蛋白(hK'-Fc)。采用PCR扩增hK基因和IgG1的Fc序列,用鼠源信号肽序列替换hK基因原有的信号肽序列,构建改良型融合蛋白hK'-Fc以及天然型融合蛋白hK-Fc的表达载体,转染中国仓鼠卵巢细胞(CHO)细胞,筛选稳定分泌融合蛋白的细胞株,通过Western blotting鉴定信号肽改造效果,利用Protein A+G亲合层析柱纯化融合蛋白,酶学实验检测融合蛋白的体外活性。结果表明:成功构建了pcDNA-hK'-Fc以及pcDNA-hK-Fc重组表达载体;获得了稳定表达融合蛋白的细胞株,产量达11mg/L以上;信号肽改造后融合蛋白的分泌效率提高约5~10倍;融合蛋白能水解其特异性的底物S-2266,具有生物学活性。本研究为进一步探讨融合蛋白的体内半衰期打下了坚实基础,也为研制治疗脑梗塞疗效更好的第二代hK蛋白和其他药用蛋白的改良提供新的线索。 相似文献
17.
Specific chemical cleavage of human placental and porcine muscle glucosephosphate isomerases at three amino peptide bonds of cysteinyl residues with 2-nitro-5-thiocyanobenzoic acid was achieved. Four primary peptides were generated from the cyanylated human glucosephosphate isomerase, indicating the quantitative cleavage of this enzyme. Four primary plus six overlap peptides were obtained from the cleavage of the swine muscle enzyme. The peptides were separated by SDS-polyacrylamide gel electrophoresis and eluted from the gels. Amino acid and carboxyl terminal analyses of the eluted peptides have permitted the alignment of these peptides with respect to the native polypeptide chain. The analysis of the enzyme which had been specifically covalently labeled at the essential lysine and histidine residues of the active center revealed that the active-site histidine and lysine residues are located on two distinct peptides with molecular weights of 27,500 and 14,000, respectively. 相似文献
18.
Oka C Tanaka M Muraki M Harata K Suzuki K Jigami Y 《Bioscience, biotechnology, and biochemistry》1999,63(11):1977-1983
To get high level secretion of human lysozyme in Pichia pastoris, the following three signal sequences and one prepro sequence were evaluated: chicken lysozyme signal peptide, leucine-rich artificial signal peptide, Saccharomyces invertase signal peptide, and Saccharomyces prepro sequence of alpha factor (MF-alpha Prepro). Transformants harboring a lysozyme gene with MF-alpha Prepro secreted 20-fold more lysozyme than those harboring the lysozyme gene with any one of the other three signal sequences. Three mutant leader sequences derived from MF-alpha Prepro were constructed to discover the function of the pro region. The secretion was dramatically decreased by eliminating the pro region of MF-alpha Prepro. In contrast, MF-alpha Prepro with the EAEAEA sequence directed the secretion of an equivalent level of lysozyme having the extra amino acids (EAEAEA) in its N-terminus. For the effective secretion of native human lysozyme, MF-alpha Prepro without any spacer sequences was most suitable. The secreted protein by MF-alpha Prepro construct was identical with the authentic human lysozyme, judging from N-terminal amino acid sequencing and molecular mass spectrometric and crystallographic analysis. 相似文献
19.
Anjali V. Sahasrabudhe Suresh M. Solapure Rajeev Khurana Vepa Suryanarayan Sudha Ravishankar Sunita M. deSousa Goutam Das 《Protein expression and purification》1998,14(3):425-433
hBSSL and its truncated variant hBSSL-C cDNA clones were expressed inPichia pastorisusing two different signal peptides, native signal peptide and invertase signal peptide, respectively, to facilitate secretion of the recombinant proteins into the culture medium. Both recombinant proteins were secreted into the culture medium to a level of 45–50 mg/liter in shake flask cultures. Native signal peptide of hBSSL was recognized inP. pastorisand was cleaved at the same site as in humans. The level of expression of the hBSSL gene was found to be dependent on the number of its copies integrated into the host chromosome. The multicopy transformant clone was found to be very stable. When grown and induced in a fermentor, the level of accumulation of the recombinant hBSSL in the culture medium improved from 50 mg/liter in shake flask cultures to 300 mg/liter. The recombinant hBSSL purified from the culture supernatant was found to be similar to the native hBSSL in its biochemical properties except for the lectin-binding profile. 相似文献
20.
Soybean lipoxygenase 1 was studied using limited proteolysis and active-site labeling to begin the structural characterization of the enzyme in solution. The serine proteases trypsin and chymotrypsin cleaved the large monomeric protein (95 kDa) into two large polypeptides, a C-terminal fragment of about 30 kDa and an N-terminal fragment of about 60 kDa. Under conditions that led to complete cleavage of the protein as judged by SDS-polyacrylamide gel electrophoresis, the catalytic activity of the protein was either reduced slightly (chymotrypsin) or enhanced (trypsin). The characteristics of the cleaved enzymes were the same as for native lipoxygenase 1 in all aspects examined: insensitivity to cyanide, fluoride, and EDTA, regiochemical and stereochemical consequences of catalysis, and EPR spectroscopy upon oxidation by product. The two fragments apparently were tightly associated as they could not be resolved under conditions which preserved the catalytic activity. Both native and protease-cleaved lipoxygenase 1 formed covalent adducts when treated with either 14C-phenylhydrazine or 4-nitrophenylhydrazine. The label was found only in the 60-kDa fragment and following complete trypsin digestion was associated with a peptide beginning after Lys-482 in the primary sequence. Therefore labeling occurred in the vicinity of the conserved histidine cluster which has been postulated as the iron-binding site. From these observations it appears that lipoxygenase 1 exists as a pair of tightly associated domains with the iron-binding site located in the larger of the two. 相似文献