重组刺桐胰蛋白酶抑制剂a的制备及其在tPA突变体纯化中的应用

将基因工程菌株E.coliBL21(DE3) pET22b-mETIa高密度发酵,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导,重组刺桐胰蛋白酶抑制剂a(rETIa)蛋白在E.coli中得到较高水平表达,表达量占菌体总蛋白的40%以上.经菌体破碎、包涵体变性、复性,二步柱层析纯化得到电泳纯的rETIa蛋白.测得rETIa对t-PA突变体(NTA)的抑制平衡常数K_i为8.72×10^－8 mol/L.据此利用纯化的rETIa蛋白制备rETIa-Sepharose 4B亲和层析柱.直接一步纯化NTA复性液,纯化的NTA纯度达90 %以上,收率为96.2 %,纯化倍数为13.2,比活为(565.7±71.3) U/μg.     

地黄GGPPS1基因克隆及表达分析

以地黄为材料,通过分析地黄转录组数据,设计特异性引物,克隆了地黄牻牛儿基牻牛儿基焦磷酸合酶（geranylgeranyl pyrophosphate synthase,GGPPS）基因的cDNA序列,命名为RgGGPPS1,GenBank登录号为KU258808。同时在生物信息学分析的基础上,进行原核表达、纯化以及组织特异性表达分析。结果显示：（1）RgGGPPS1基因开放阅读框为987 bp,编码328个氨基酸。（2）生物信息学分析结果显示,RgGGPPS1蛋白含有2个富含天冬氨酸的基序(DDXXXXDD和DDXXD),与芝麻等双子叶植物中的GGPPS蛋白相似性较高。（3）利用构建的原核表达载体pET 32a RgGGPPS1在大肠杆菌BL21(DE3)菌株中成功表达RgGGPPS1重组蛋白,采用Ni²⁺亲和层析得到了纯化的RgGGPPS1重组蛋白。（4）荧光定量PCR结果显示,RgGGPPS1基因在根中表达量最高,叶、茎中表达量较低。研究结果为进一步研究RgGGPPS1基因在地黄环烯醚萜苷生物合成途径中的功能奠定了基础。     

HIV-1 Vpu蛋白的原核表达、鉴定和纯化

目的：克隆、表达、纯化人免疫缺陷病毒Ⅰ型（HIV-1）Vpu蛋白,为其功能及免疫学研究奠定基础。方法：PCR扩增Vpu基因,纯化、酶切后克隆到原核表达载体pET32a中,转化大肠杆菌BL21（DE3）菌株获得表达工程菌株,IPTG诱导蛋白表达,免疫印迹鉴定目的蛋白,亲和层析纯化蛋白。结果：构建了HIV-1Vpu蛋白的原核表达载体Vpu-pET32a,并在大肠杆菌中高效表达,目的蛋白呈可溶性形式存在,免疫印迹检测显示为目的蛋白,经Ni—NTAAgarose纯化获得了高纯度的目的蛋白。结论：在原核表达系统中表达了可溶性HIV-1Vpu蛋白,为进一步进行HIV-1Vpu蛋白的免疫原性和功能研究奠定了基础。     

一株MAs (III)脱甲基菌株的分离及功能基因的研究

[目的] 分离并鉴定三价单甲基砷（MAs （III））脱甲基菌株,对MAs （III）脱甲基菌FJ-6中arsI基因进行克隆表达,并对arsI基因表达蛋白进行功能鉴定。[方法] 利用富集培养的方法分离MAs （III）脱甲基菌株,并通过形态学、生理生化特征和16S rDNA基因进化分析进行鉴定;HPLC-ICP-MS鉴定菌株转化MAs （III）的产物为三价砷（As （III））,对菌株FJ-6的基因组进行生物信息学分析,寻找潜在的MAs （III）脱甲基酶编码基因,通过PCR扩增获得arsI全长基因,构建重组质粒pET29a-arsI,转化大肠杆菌BL21（DE3）菌株进行异源表达,通过Ni²⁺-NTA亲和层析柱纯化异源表达的蛋白,以MAs （III）为反应底物,检测MAs （III）脱甲基酶ArsI的酶学特性。通过实时定量PCR观察arsI的表达类型。[结果] Bacillus aryabhattai FJ-6在12 h内能将1 μmol/L MAs （III）完全转化为As （III）。克隆得到MAs （III）脱甲基酶表达基因arsI,构建了pET29a-arsI重组质粒并进行了表达,ArsI蛋白分子量为17.4 kDa。ArsI纯化蛋白具有较高的MAs （III）脱甲基酶的活性;荧光定量PCR实验结果表明arsI受砷诱导表达。[结论] 明确了ArsI蛋白具有MAs （III）脱甲基酶活性。     

红细胞嘧啶-5′-核苷酸酶的纯化及其抗血清的制备

利用UMP-ADH-Sepharose4B亲和层析的方法纯化正常人红细胞嘧啶-5′-核苷酸酶（P5′N, EC.3.1.3.5）.结果表明：利用UMP-ADH-Sepharose4B亲和层析柱可有效、专一性吸附P5′N,纯化液经聚丙烯酰胺凝胶电泳显示一条蛋白质带,测定纯化物相对分子质量28×10³,等电点（pI）5.2. 纯化物免疫家兔后,得兔抗人P5′N抗血清,为探讨遗传性及获得性P5′N缺陷奠定了基础.     

人源脂肪酸合酶在酿酒酵母中的表达纯化和结构的初步分析

[背景] 聚酮类化合物在医药领域有重要的应用,相关药物研发依赖聚酮合酶多变的结构认知,人源脂肪酸合酶的组成结构和催化机制与聚酮合酶相近,研究人源脂肪酸合酶结构可为聚酮合酶的研究奠定基础。[目的] 在酿酒酵母中表达纯化人源脂肪酸合酶蛋白,确定合适的体外纯化条件。[方法] 以酿酒酵母BJ5464为表达载体,构建带有His和Strep双亲和层析标签的重组质粒,诱导表达蛋白后用亲和层析方法获取目标蛋白,并结合凝胶电泳和快速蛋白质液相层析技术,确定合适的蛋白纯化条件。[结果] 成功构建重组表达质粒pxw55-hfas-cSHII, 并在体外纯化得到合适浓度和纯度的人源脂肪酸合酶蛋白,筛选不同缓冲液条件并结合电子显微镜观察结果反馈,确定合适的蛋白体外纯化体系。[结论] 蛋白电镜结构分析需要有高纯度、合适浓度并且形成正确构象的蛋白样品,而人源脂肪酸合酶蛋白纯化体系的建立和纯化条件的确定为其电镜结构分析提供了良好的样品,为人源脂肪酸合酶的结构解析及结构相似但更为复杂的聚酮合酶蛋白解析奠定了良好基础。     

人胱硫醚β合成酶原核表达纯化及鉴定

目的：构建人胱硫醚β合成酶（human cystathionineβ-synthase,hCBS）基因原核表达载体,在E.coli BL21（DE3）中表达,并进行纯化和酶活性检测。方法：以胰腺细胞cDNA文库为模板,采用聚合酶链式反应（PCR）扩增hCBS基因蛋白编码区的全序列,克隆入原核表达载体pET32a（＋）,构建重组质粒pET32a（＋）-hCBS。经限制性内切酶双酶切及DNA序列分析鉴定目的基因后与人CBS基因（基因bank号：BT007154.1）完全一致,转入E.coli BL21（DE3）中,由IPTG诱导表达融合蛋白。结果：经SDS-PAGE、Western blot分析,证明诱导表达的蛋白为重组人CBS（rhCBS）。再由Ni-NTA树脂亲和层析,并脱盐冷冻干燥后获得重组rhCBS（约19 mg/L培养物）,并测得其比活力约为57 kU/g。结论：成功地表达纯化出具有功能活性的重组蛋白rhCBS,为进一步研究该酶的相互作用蛋白以及其在生物学和临床科学的作用奠定了基础。     

人血液血管细胞生成素的胎肝免疫印记检测

目的：制备一种新蛋白——人血液血管细胞生成素（hemangiopoietin,HAPO）的单克隆抗体,检测其在胎肝中的表达。方法：杂交瘤方法制备抗HAP0的单克隆抗体;非竞争酶联免疫吸附实验测定抗体的相对亲和力;蛋白G亲和层析纯化腹水中的抗体,免疫印记方法检测胎肝中天然HAPO的表达：结果：所获单抗分别为IgG1及IgM,其轻链均为κ。三株IgG1亚类单抗的相对亲和力分别为3．06×10^9mol／L,6．07×10^8mol／L和1．71×10^10 mol／L。亲和纯化后抗体的纯度达99％以上：胎肝中在蛋白水平上可以检测到HAP0的表达,天然HAPO的分子量接近于重组HAPO的分子量。结论：人胚胎肝组织中在蛋白水平上可以检测到HAPO的表达.     

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.     

HCMV IE86蛋白与转录激活因子的相互作用

将人巨细胞病毒IE86 cDNA克隆入pGEX-2T表达载体, 在大肠杆菌中表达出GST-IE86融合蛋白. SDS-PAGE和Western blot分析表明, GST-IE86融合蛋白存在于细胞裂解上清中, 为可溶性蛋白, 分子量为92 ku. 通过亲和层析柱纯化, 分别获得纯化的GST和GST-IE86融合蛋白. 采用特异性亲和层析共分离技术研究HCMV IE86 蛋白与转录调控蛋白及转录因子相互作用, 表明IE86能与直接结合启动子DNA的转录激活因子SP1, AP1, AP2及转录因子TFⅡB相互作用而形成异源蛋白复合物. 该转录激活因子及转录因子与IE86蛋白同时被吸留在亲和层析柱中, 但IE86蛋白不能与NF-κB相互作用. 提示IE86蛋白与转录激活因子及转录因子相互作用的活性与IE86蛋白的糖基化无关;IE86蛋白激活多种基因转录是由于IE86蛋白具有两个蛋白质结合位点, 它们与激活转录的调控蛋白和转录因子结合, 这种相互作用加速了转录起始复合物的装配.     

Homonyms, synonyms and mutations of the sequence/structure vocabulary

The effect of pH and temperature on the association equilibrium constant (Ka) for bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B has been investigated. Ka values decrease with decreasing pH, reflecting the acid-midpoint and pK shifts, upon BPTI binding, of a three-proton co-operative transition, between pH 3 and 5, and of a single ionizable group, between pH 5 and 9. At pH 8, the values of delta H degree (between 7 degrees C and 42 degrees C) and delta S degree (at 21 degrees C) for BPTI binding to the glandular kallikreins considered were determined. In particular, the delta H degree values have been found to be independent of temperature and the following values have been obtained by van't Hoff plots: +1.8 kcal/mol, +2.3 kcal/mol and +2.4 kcal/mol (1 kcal = 4184 J) for the inhibitor binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B, respectively. Considering the known molecular structures of free porcine pancreatic beta-kallikrein A and BPTI, and of their complex, the stereochemistry of the enzyme : inhibitor contact regions was analysed for the three serine proteinases, in relation to their respective types of behaviour.     

Primary structure of human urinary prokallikrein

The complete amino acid sequence of human urinary prokallikrein has been determined by amino acid analysis and sequence determination of peptide fragments obtained from chemical and enzymological cleavages of kallikrein and by comparison of the N-terminal sequence of prokallikrein with that of kallikrein, the active form. Prokallikrein was a single chain polypeptide which comprised 238 amino acid residues of kallikrein and 7 amino acid residues of the propeptide. The sequence, Asn-X-Thr(Ser), which is a common glycosylation site was found at positions 78-80, 84-86, and 141-143. Two trypsin-susceptible sites were identified. One is the Arg(-1)-Ile(1) bond and the other is the Arg (87)-Gln(88) bond. The sequence of human urinary kallikrein was identical with that of human pancreatic and kidney kallikreins (Fukushima, D. et al. (1985) Biochemistry 24, 8037-8043; Baker, A.R. & Shine, J. (1985) DNA 4, 445-459), which were predicted from the nucleotide sequences of cDNAs. The primary structure of human urinary kallikrein is homologous to those of the other animal kallikreins and kallikrein-related proteins. Key amino acid residues, His(41), Asp(96), and Ser(190), required for catalytic activity and Asp (184) required for kallikrein-type specificity are completely conserved. The results show that human urinary prokallikrein and kallikrein are of tissue type and they are excreted in urine without any modification.     

Inhibition of Serine Proteinases by P-Carbethoxyphenyl Esters of ϵ-Guanidino- And ϵ-Amino Caproic Acid: Thermodynamic and Molecular Modeling Study

Abstract

The inhibitory effect of the clinically used p-carbethoxyphenyl ester of ?-guanidino-caproic acid metha-nesulphonate (?-GCA-CEP) on the catalytic properties of human LYS⁷⁷-plasmin (EC 3.4.21.7), bovine factor Xa (EC 3.4.21.6), bovine α-thrombin (EC 3.4.21.5), ancrod (EC 3.4.21.28), crotalase (EC 3.4.21.30), bovine β-trypsin (EC 3.4.21.4), porcine pancreatic β-kallikrein-B (EC 3.4.21.39, human urinary kallikrein (EC 3.4.21.35) and the M_r 54,000 species of human urokinase (EC 3.4.21.31) was investigated (between pH 2.0 and 8.5, I = 0.1 M;T = 21 ? 0.5?C), and analyzed in parallel with that of the homologous derivative p-carbethoxyphenyl ?-amino-caproate hydro chloride (?-ACA-CEP). On lowering the pH from 5.5 to 3.0, values of the apparent dissociation inhibition constant (K_i) for ?-GCA. CEP and ?-ACA-CEP interaction with the serine proteinases considered increase, reflecting the acidic pK-shift upon inhibitor binding of a single ionizing group. Over the whole pH range explored, (i) ?-GCA-CEP interacts with bovine factor Xa and bovine α-thrombin with an higher affinity than that observed for ?-ACA-CEP binding; (ii) both inhibitors associate to bovine β-trypsin with the same affinity; and (iii) ?-ACA-CEP inhibits human Lys⁷⁷-plasmin and the M_r 54,000 species of human urokinase with an higher affinity than that reported for ?-GCA-CEP association, thus reflecting the known enzyme primary specificity properties. However, the affinity of ?-ACA-CEP for ancrod, crotalase, porcine pancreatic β-kallikrein-B and human urinary kallikrein, all of which preferably bind arginyl rather than lysyl side chains at the primary position of substrates and/or inhibitors, is paradoxically higher than that displayed by ?-GCA-CEP. By considering the amino acid sequences, the X-ray three-dimensional structures and/or the computer-generated molecular models of serine proteinase: inhibitor adducts, the observed binding behaviour of ?-GCA-CEP and ?-ACA-CEP to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).     

Crystal structure of recombinant human tissue kallikrein at 2.0 A resolution.

Human tissue kallikrein, a trypsin-like serine protease involved in blood pressure regulation and inflammation processes, was expressed in a deglycosylated form at high levels in Pichia pastoris, purified, and crystallized. The crystal structure at 2.0 A resolution is described and compared with that of porcine kallikrein and of other trypsin-like proteases. The active and S1 sites (nomenclature of Schechter I, Berger A, 1967, Biochem Biophys Res Commun 27:157-162) are similar to those of porcine kallikrein. Compared to trypsin, the S1 site is enlarged owing to the insertion of an additional residue, cis-Pro 219. The replacement Tyr 228 --> Ala further enlarges the S1 pocket. However, the replacement of Gly 226 in trypsin with Ser in human tissue kallikrein restricts accessibility of substrates and inhibitors to Asp 189 at the base of the S1 pocket; there is a hydrogen bond between O delta1Asp189 and O gammaSer226. These changes in the architecture of the S1 site perturb the binding of inhibitors or substrates from the modes determined or inferred for trypsin. The crystal structure gives insight into the structural differences responsible for changes in specificity in human tissue kallikrein compared with other trypsin-like proteases, and into the structural basis for the unusual specificity of human tissue kallikrein in cleaving both an Arg-Ser and a Met-Lys peptide bond in its natural protein substrate, kininogen. A Zn+2-dependent, small-molecule competitive inhibitor of kallikrein (Ki = 3.3 microM) has been identified and the bound structure modeled to guide drug design.     

Expression and Characterization of Human Tissue Kallikrein Variants

Human tissue kallikrein is a serine protease implicated in the pathology of various inflammatory disorders. As one of the two principal enzymes that generate proinflammatory kinin peptidesin vivo,tissue kallikrein represents an attractive target for therapeutic intervention in diseases such as asthma, pancreatitis, and rheumatoid arthritis. Three distinct human tissue kallikrein variants, differing in one or two amino acid substitutions, are predicted to exist based on genomic or cDNA nucleotide sequences derived from different tissues. The effects of these substitutions on the biochemical properties of tissue kallikrein are unknown but could, in principle, confer tissue-specific functions on the enzyme or affect the clinical utility of specific kallikrein inhibitors. All three variants, as well as a deglycosylated derivative, were expressed in high yield as recombinant proteins inPichia pastoris.The recombinant kallikrein variants and natural urinary kallikrein all hydrolyzed synthetic peptides with similar specificity and efficiency and released kallidin from kininogen at comparable rates. Similarly, no significant differences were observed in the interactions between kallikrein variants and protein inhibitors such as SBTI, α₁-PI, and aprotinin. We conclude that the known tissue kallikrein variants represent allelic variants and are not likely to have tissue-specific activity related to the amino acid substitutions.     

Purification of single-chain human low-molecular-weight kininogen and demonstration of its cleavage by human urinary kallikrein

Human low-molecular-weight kininogen (LMWK) was purified to apparent physical and functional homogeneity by a six-step procedure consisting of ion-exchange chromatography, reverse ammonium sulfate gradient solubilization, hydrophobic chromatography on phenyl-Sepharose, gel filtration, and removal of contaminating proteins by their affinity for Affi-Gel blue and zinc. The recovery averaged 15.6% (n = 4). Purified LMWK presented as a single stained band on alkaline polyacrylamide gel electrophoresis which corresponded to the region of function in eluates from a duplicate gel. The apparent homogeneity was also observed in sodium dodecyl sulfate (SDS)-gel electrophoresis, where the protein presented as a single band of Mr = 65,000 without reduction and 68,000 with reduction. A mole of substrate released 0.8 mol of kinin in 5 min when cleaved by human urinary kallikrein (HUK), and 0.9 mol after 30 min. Cleavage of the single-chain LMWK released kinin from within a disulfide loop as indicated by the SDS-gel electrophoresis of reduced and unreduced kinin-free LMWK. The heavy chain exhibited an Mr = 62,000, which is similar to the Mr of the amino-terminal chain of human HMWK and is consistent with their antigenic relatedness. In contrast to the Mr = 64,000 procoagulant chain of human HMWK, the small (less than 10,000) carboxy-terminal chain of LMWK has no procoagulant activity and may serve only to protect the kinin moiety in the intact substrate.     

Similarity between a kininogenase (kallikrein) from human large intestine and human urinary kallikrein.

A human colon kininogenase (kallikrein) was isolated by gel filtration on Sephacryl S-200 and affinity chromatography on Trasylolbound Sepharose, yielding a material with a specific activity of 1.3 U/mg (substrate: AcPheArgOEt). The molecular weight of the enzyme as estimated by gel filtration is approximately 70 000. After reduction with mercaptoethanol two bands were obtained in dodecyl sulfate eletrophoresis with molecular weights of 27 000 and 70 000. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 4 l x mol-1 x min-1. The preparation was characterized by immunological and enzymatic methods. Using the radioimmumoassay for human urinary kallikrein cross-reactivity and parallel binding curves were obtained. Kinin liberation from human high Mr-kininogen was totally inhibited by antibodies directed against human urinary kallikrein. Trasylol and diisopropyl fluorophosphate, but not by antibodies directed against human trypsin and plasma kallikrein. The effect on dog blood pressure was comparable to that obtained with human urinary kallikrein. The amino acid composition of human large intestine kallikrein is very similar to that of human urinary kallikrein.     

Crystal structure of human epidermal kallikrein 7 (hK7) synthesized directly in its native state in E. coli: insights into the atomic basis of its inhibition by LEKTI domain 6 (LD6)

Human kallikrein 7, a major protease of human skin, has been synthesized directly in its native conformation in Escherichia coli by forcing the secretion of the newly synthesized polypeptide into the bacterial periplasm. The procedure yields a stable kallikrein 7 with highly specific activity that is inhibited efficiently by its specific inhibitor LEKTI domain 6. The protein was crystallized, and its three-dimensional structure was solved in the absence of protease inhibitors. The structure obtained agrees with that reported recently for human tissue kallikrein 7 crystallized in the presence of protease inhibitors from a preparation obtained in a baculovirus protein expression system. A model of the interaction between the protease and its inhibitor is proposed on the basis of both the three-dimensional structure of human tissue kallikrein 7 reported here and that of the LEKTI domain 6 solved previously by NMR.     

Sequence of a new Bowman-Birk inhibitor fromTorresea acreana seeds and comparison withTorresea cearensis trypsin inhibitor (TcTI2)

TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds ofTorresea acreana, one of the two known species ofTorresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears asM _r 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2,Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.     

Isolation of tissue kallikrein in rat spleen by monoclonal antibody-affinity chromatography

Rat spleen kallikrein was identified and purified by DEAE-cellulose and monoclonal antibody-affinity chromatography. The purified enzyme has Tos-Arg-OMe esterase activity and kinin-releasing activity from a purified low-molecular-weight kininogen substrate. In the direct radioimmunoassay for tissue kallikrein, the splenic enzyme displays parallelism with standard curves of rat urinary kallikrein. The pH profiles of the Tos-Arg-OMe esterase activities of spleen and urinary kallikrein were identical with optima at 9.0 Rat spleen kallikrein was inhibited strongly by aprotinin and affinity-purified kallikrein antibody and weakly by soybean trypsin inhibitor. The IC₅₀ values were similar to those observed against rat urinary kallikrein. Neither the urinary nor the splenic enzyme was inhibited by lima bean trypsin inhibitor or preimmune serum immunoglobulins. Spleen kallikrein was labeled with [¹⁴]diisopropylphosphorofluoridate and visualized by fluorography on a sodium dodecyl sulfate-polyacrylamide gel. The electrophoretic mobility of the splenic enzyme was indistinguishable from that of urinary kallikrein A with an estimated Mr of approx. 38 000. With Western blot analyses using a rabbit anti-kallikrein antibody followed by ¹²⁵I-labeled protein A binding, the spleen and urinary kallikreins were again visualized at identical positions by autoradiography. The data show that there is a rat splenic tissue kallikrein which is indistinguishable from a renal kallikrein with respect to physicochemical properties, immunological character and susceptibility to inhibitors.