Immunoadsorbent Chromatography Research Articles

Characterization of Alpha-Fetoprotein in Fetal Striped Dolphin (Stenella coeruleoalba): Purification of Protein Product and Molecular Cloning of the Corresponding Transcript

Alpha-fetoprotein (AFP) is a fetal glycoprotein that is known as a biomarker for monitoring pregnancy in many mammalian species. However, characterization of AFP has not yet been undertaken in any cetacean species. Here, we purified AFP from the serum of fetal striped dolphin by chemical precipitation followed by a combination of immunoadsorbent column chromatography and gel filtration. The molecular masses of native and denatured dolphin AFP were estimated to be ∼78,000 Da by gel filtration and ∼68,000 Da by SDS-PAGE, respectively, representing typical masses reported for mammalian AFPs. In fetal serum, only the AFP band (∼68,000 Da) appeared to be immunoreactive to an antiserum against purified dolphin AFP, indicating sufficient specificity for the development of an AFP immunoassay. Full-length cDNA encoding for the dolphin AFP was cloned from fetal liver and revealed an open reading frame comprising 610 amino acid residues, which included a putative signal peptide of 18 amino acid residues. This was followed by a sequence identical to the N-terminus of purified AFP. The deduced amino acid sequence of dolphin AFP showed more than 80% identity to those of other mammalian AFPs. To our knowledge, the present report represents the first identification and characterization of AFP from any cetacean species.

Purification and Classification of Three Lipovitellin Subtypes in the Marbled Sole (Pleuronectes yokohamae)

An immunologlcal analysis using subtype-specific antisera of the major yolk protein lipovltellin (Lv) of the grey mullet (Mugil cephalus) confirmed the presence of the three corresponding Lv subtypes (LvA, LvB, and LvC) in vitellogenic ovaries of the marbled sole (Pleuronectes yokohamae). These three Lv subtypes were purified from sole ovaries by using various combinations of anion exchange, hydroxylapatite, immunoadsorbent, and gel-filtration chromatography. Purified LvA, LvB, and LvC had an apparent native mass of approximately 482, approximately 380, and approximately 372 kDa, respectively, estimated by gel filtration. Analysis of their tertiary structures by SDS-PAGE indicated that LvA, LvB, and LvC were typical of teleost Lvs in having a heavy (H) chain (approximately 105, approximately 102, and approximately 107 kDa, respectively) and a light (L) chain (approximately 22, approximately 19.5, and approximately 25 kDa, respectively). The N-termlnal amino acid (AA) sequences were obtained for the LvA H chain, the LvB H and L chains, and the LvC L chain and compared to the deduced AA sequences of their precursors, vitellogenins (Vgs), in several species. This comparison of LvA, LvB, and LvC with various teleost VgA, VgB, and VgC sequences, respectively, revealed high identities (60-100%). The purified Lv subtypes were subjected to double immunodiffusion using an antiserum against an unclassified Lv of the sole ( Hashimoto et al., 1998 ); only the LvB subtype exhibited immunoreactivity with this antiserum. This result indicates that the previously developed immunoassay using this anti-Lv for the detection of sole Vg is effectively a VgB-specific assay.

Glycoprotein H (gII/gp108) and glycoprotein L form a functional complex which plays a role in penetration, but not in attachment, of bovine herpesvirus 1.

The glycoproteins of bovine herpesvirus 1 (BHV-1) play important roles in the interactions between virions and target cells. A 108 kDa glycoprotein, designated gII or gp 108, has been identified by two different panels of monoclonal antibodies. The gII- and gp 108-specific monoclonal antibodies were shown to react with the same protein, which was identified by N-terminal sequencing as the homologue of herpes simplex virus type 1 (HSV-1) gH. When BHV-1 gH was purified by immunoadsorbent chromatography, gL was co-purified. The gH-gL complex induced the production of antibodies that neutralized virus infectivity and inhibited virus penetration. Affinity-purified gH-gL did prevent penetration, but not attachment of BHV-1, which suggests that the gH-gL complex is essential for penetration of BHV-1 into susceptible cells.

Several Closely Related Glutathione S-Transferase Isozymes Catalyzing Conjugation of 4-Hydroxynonenal Are Differentially Expressed in Human Tissues

A human acidic glutathione S-transferase, hGST 5.8, was isolated from heart, pancreas, and brain by a procedure involving immunoadsorption chromatography on immobilized antibodies raised against mouse mGSTA4-4. The human hGST 5.8 enzymes isolated from these tissues had similar pI (5.8) and subunit Mr (24.5 kDa) values, showed about 17- to 20-fold higher specific activities for 4-hydroxynon-2-enal than that for 1-chloro-2 4-dinitrobenzene, and expressed glutathione peroxidase activity toward phospholipid hydroperoxides. In this respect, the enzymes belong together with rat GST 8-8 and mouse mGSTA4-4 to a subgroup of GSTs involved in the detoxification of lipid peroxidation products. Partial sequencing of CNBr-peptide fragments of hGST 5.8 proteins isolated from various human tissues revealed significant similarity to mGSTA4-4 and the existence of several distinct isoforms differing in their primary structures. These isoforms had similar but nevertheless clearly distinguishable catalytic properties. These results indicate the existence of multiple hGST 5.8-related genes in the humans, which is consistent with our previous studies showing the presence of several closely related genes for the mouse ortholog mGSTA4-4 (Zimniak et al., J. Biol. Chem., 1994, 269, 992-1000).

Folding of the four domains and dimerization are impaired by the Gly446.fwdarw.Glu exchange in human glutathione reductase. Implications for the design of antiparasitic drugs

Glutathione reductase (NADPH+GSSG+H+-->NADP(+) + 2GSH) is a homodimeric flavoenzyme of known geometry. Each subunit contains four well-defined domains and contributes essential residues to the active sites; consequently, the monomer is expected to be inactive. As part of our program to develop dimerization inhibitors of human glutathione reductase (hGR) as antimalarial agents, we mutagenized the residues 446 and 447 which, together with their counterparts on the other subunit, represent the tightest contact between the subunits [Karplus, P. A., & Schulz, G. E. (1987) J. Mol. Biol. 195, 701-729]. Wild-type human glutathione reductase and mutants of this protein were produced in plasmid-transformed Escherichia coli SG5 cells. Active enzyme species, namely, wild-type hGR, N-terminally truncated delta(1-15)hGR, and the point mutant F447P-hGR, were purified by 2',5'-ADP-Sepharose chromatography and crystallization. Inactive mutants such as G446E-hGR or the double mutants G446E/F447P-hGR and G446P/F447P-hGR were isolated by immunoadsorption chromatography. G446E/F447P-hGR was studied in detail. This mutant behaved like a poorly folded monomeric protein, as indicated by the following properties: absence of the intersubunit disulfide bridge, Cys90-Cys90'; failure to bind FAD; failure to bind NADPH and analogues thereof; a short half-life (< 4 min) in E. coli cells; and high susceptibility to trypsin in vitro. The results suggest that the sequence around G446 can control dimerization as well as domain folding. This is unexpected since the FAD-binding domain and the NADPH-binding domain occur in many different enzymes and have been regarded as autonomous folding units.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile.

This study was undertaken to examine toxin production by Clostridium difficile 8864, a naturally occurring isolate that has been reported to produce toxin B in the absence of toxin A. To date, this is the only strain of C. difficile reported to produce only one of the toxins. The results of our initial studies with antibodies against toxins A and B confirmed these observations. Toxin B from strain 8864 and from VPI strain 10463, a strain that produces high levels of both toxin A and toxin B, was purified to homogeneity by sequential anion-exchange chromatography on DEAE-Sepharose CL-6B, gel filtration on Ultrogel AcA22, and immunoadsorption chromatography, and their toxic activities were compared. Our results showed that toxin B from strain 8864 and toxin B from C. difficile VPI strain 10463 were comparable in their cytotoxic activities and that the 8864 toxin B was more lethal. In addition, we observed that toxin B from strain 8864 was weakly enterotoxic, which may explain the ability of this strain to cause intestinal disease in hamsters treated with antibiotics. Analysis with specific antibodies showed that the toxin B molecules from these strains were highly related but contained distinct epitopes. The results of hybridization studies with probes specific for the toxin B gene of VPI strain 10463 demonstrated differences between the toxin B genes of the two strains. In addition, probes specific for the toxin A gene of VPI strain 10463 showed that strain 8864 contains a region which shows identity with the 5' end of the toxin A gene but not the region of the gene which encodes a hydrophobic region and the repeating units.

Location of the two catalytic sites in intestinal lactase-phlorizin hydrolase. Comparison with sucrase-isomaltase and with other glycosidases, the membrane anchor of lactase-phlorizin hydrolase.

Lactase-phlorizin hydrolase was isolated by immunoadsorption chromatography from rabbit brush-border membrane vesicles. Inactivation of the enzyme with [3H]conduritol-B-epoxide, a covalent active site-directed inhibitor, labeled glutamates at positions 1271 and 1747. Glu1271 was assigned to lactase, Glu1747 to phlorizin hydrolase activity. In contrast, the nucleophiles in the active sites of sucrase-isomaltase are aspartates (Asp505 and Asp1394). Asp505 is a part of the isomaltase active site and is localized on the larger subunit, which carries the membrane anchor also, while Asp1394 is a part of the active of sucrase. Alignment of these 2 nucleophilic Glu residues in lactase-phlorizin hydrolase and of their flanking regions with published sequences of several other beta-glycosidases allows the classification of the configuration retaining glycosidases into two major families: the "Asp" and the "Glu" glycosidases, depending on the carboxylate presumed to interact with the putative oxocarbonium ion in the transition state. We offer some predictions as to the Glu acting as the nucleophile in the active site of some glycosidases. By hydrophobic photolabeling, the membrane-spanning domain of lactase-phlorizin hydrolase was directly localized in the carboxyl-terminal region thus confirming this enzyme as a monotopic type I protein (i.e. with Nout-Cin orientation) of the brush-border membranes. A simplified version of the Me2+ precipitation method to efficiently and simply prepare brush-border membrane vesicles is also reported.

Partial purification of horse-specific soluble muscle proteins by immunoadsorption chromatography

Immunoadsorption chromatography has been used to isolate horse-specific soluble muscle proteins from crude horse protein extracts. Horse-specific polyclonal antibodies against soluble horse muscle proteins immobilised on a Protein A-Sepharose CL-4B matrix were used to adsorb the corresponding antigens. Analysis of the crude soluble horse muscle proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed the existence of 20 protein subunits in the gel. SDS-PAGE of the proteins recovered by affinity chromatography showed nine protein subunits, three of which were enriched after the immunopurification step. These affinity-recovered horse-specific soluble muscle proteins may be used as antigens in the development of monoclonal antibodies to detect and quantify horse meat in raw, unheated meat mixtures.

Purification and partial characterization of branched-chain α-ketoacid dehydrogenase kinase from rat liver and rat heart

Branched-chain α-ketoacid dehydrogenase kinase was purified to homogeneity from rat liver and rat heart. The initial step was the purification of rat liver and heart branched-chain α-ketoacid dehydrogenase complex with high kinase activity by a modification of a method described previously. Preservation of high kinase activity during purification of the complex required the presence of fresh dithiothreitol throughout the procedure. The kinase was released from the complex by oxidation of dithiothreitol with potassium ferricyanide and purified by high-speed centrifugation, immunoadsorption chromatography, and DEAE-Sephacel chromatography. Both kinase preparations gave only one polypeptide band with a molecular weight of 44,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphorylation and inactivation of the branched-chain α-ketoacid dehydrogenase complex by the purified kinase was inhibited by α-chloroisocaproate and dichloroacetate, established inhibitors of the phosphorylation of the branched-chain α-ketoacid dehydrogenase complex. The kinase did not exhibit autophosphorylation and does not correspond to the same protein as pyruvate dehydrogenase kinase. The kinase phosphorylated histone (type II-S), but this reaction was slow relative to the phosphorylation of the branched-chain α-ketoacid dehydrogenase complex and was not inhibited by α-chloroisocaproate.

Lymphocyte proliferative responses to separated bovine herpesvirus 1 proteins in immune cattle

The immune response to bovine herpesvirus 1 (BHV-1) infection can protect cattle from subsequent challenge with the virus. This protection involves a variety of defensive strategies, and the activation of most of these defenses requires the recognition of viral proteins by the cellular immune system. To identify some of the BHV-1 proteins recognized by T lymphocytes, we measured in vitro proliferative responses to individual proteins. Viral proteins were separated by gel electrophoresis followed by Western immunoblotting, and immunoblots were evaluated for serological reactions. Unstained blotted fractions were processed into antigen-bearing particles for analysis in blastogenesis assays. Purified BHV-1 proteins obtained by immunoadsorbent chromatography were processed and included for comparison in both enzyme-linked immunosorbent and proliferation assays. The tegument protein VP8 and the glycoprotein gIV appeared to be the antigens which most consistently stimulated the proliferation of lymphocytes from BHV-1-immunized animals. Positive blastogenic responses were also detected to gI, gIII, and to one or more uncharacterized, low-molecular-weight proteins in some of the cattle tested. These results indicate that T-lymphocyte proliferative responses to BHV-1 proteins are detectable in immune cattle and may be important in protection from BHV-1 infection.

Identification of Cysteine 530 as the Covalent Attachment Site of an Affinity-labeling Estrogen (Ketononestrol Aziridine) and Antiestrogen (Tamoxifen Aziridine) in the Human Estrogen Receptor

Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain.

A simplified method for purification of an antitumor acidic glycoprotein from Streptococcus pyogenes (Su strain) by immunoadsorbent chromatography.

A simplified method for purification of an antitumor acidic glycoprotein (SAGP) from Streptococcus pyogenes (Su strain) by immunoaffinity chromatography is described. A cell-free crude extract prepared from the cocci was applied to the anti-SAGP IgG coupled Sepharose column, and elution was conducted with an alkaline buffer. The material eluted was confirmed to be homogeneous and identical with SAGP as demonstrated by both relative mobility on the SDS-polyacrylamide gel column and the antigenicity on the double diffusion agar plate. The cell-growth inhibitory activity of SAGP prepared by the present method was almost the same as that of SAGP purified by the previous time-consuming method. Since this simplified method provides a higher yield of SAGP, it will be useful in further studies on the biological properties of SAGP.

Isolation and purification by high performance liquid chromatography of a tetanus toxin fragment (Fragment [A-B]) derived from mildly papain-treated toxin

Fragment [A-B] of tetanus toxin was highly purified by combination of gel permeation chromatography, adsorption chromatography in an HPLC and immunoadsorption chromatography using anti-Fragment [C] as a ligand. The purified Fragment [A-B] (200 μg) elicited a peculiar toxicity, ‘hypoactivity’ or ‘weakness’, and killed the mice in ca. 73 hr and 88 hr when it was injected i.v. and i.m., respectively. However, contamination by the whole toxin was not detectable, in the purified fragment preparation, when up to 600 μg was tested by the mouse toxicity assay.

Isolation of Endopeptidase-24.11 (EC 3.4.24.11, "Enkephalinase") From the Pig Stomach

Abstract The purpose of this investigation was to isolate the cell-surface enzyme endopeptidase-24.11 from the stomach wall of the pig and to examine the hydrolysis of the gastric neuropeptides. Endopeptidase-24.11 was isolated from gastric membranes by immuno-adsorbent chromatography using a monoclonal antibody to porcine kidney endopeptidase-24.11. The enzyme was purified with a yield of 1.2 μg/g wet wt of fundic muscle. A single polypeptide chain of apparent subunit molecular weight of 90,000 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gastric endopeptidase-24.11 hydrolyzed substance P, gastrin-releasing peptide 10, [Leu 5 ] enkephalin, and [Met 5 ] enkephalin by cleavage of peptide bonds on the N-terminal side of hydrophobic amino acids. The enzymatic activity was inhibited completely by phosphoramidon (10 −6 M) and strongly by 1,10-phenanthroline (10 −3 M), but was unaffected by captopril (10 −5 M).

Isolation of endopeptidase-24.11 (EC 3.4.24.11, “enkephalinase”) from the pig stomach: Hydrolysis of substance P, gastrin-releasing peptide 10, [Leu5] enkephalin, and [Met5] enkephalin

The purpose of this investigation was to isolate the cell-surface enzyme endopeptidase-24.11 from the stomach wall of the pig and to examine the hydrolysis of the gastric neuropeptides. Endopeptidase-24.11 was isolated from gastric membranes by immuno-adsorbent chromatography using a monoclonal antibody to porcine kidney endopeptidase-24.11. The enzyme was purified with a yield of 1.2 μg/g wet wt of fundic muscle. A single polypeptide chain of apparent subunit molecular weight of 90,000 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gastric endopeptidase-24.11 hydrolyzed substance P, gastrin-releasing peptide 10, [Leu5] enkephalin, and [Met5] enkephalin by cleavage of peptide bonds on the N-terminal side of hydrophobic amino acids. The enzymatic activity was inhibited completely by phosphoramidon (10−6 M) and strongly by 1,10-phenanthroline (10−3 M), but was unaffected by captopril (10−5 M).

Monoclonal antibodies specific for thiophosphorylated proteins recognize Xenopus MPF

Maturation promoting factor, (MPF), is a crucial regulatory component of the eukaryotic cell cycle. Though it is ubiquitous, MPF has been difficult to purify to homogeneity, and little is known about its physical properties or composition. In an attempt to further characterize and purify this protein, we have isolated five monoclonal antibodies that immunoadsorb MPF activity, and inhibit the activity in solution. However, all the antibodies recognize many proteins in partially purified MPF. We have shown that antibody binding is dependent on previous exposure of the preparation to ATPγS. This suggests that the antibodies specifically recognize thiophosphoproteins, although not all thiophosphorylated proteins in MPF are immunoprecipitated. Using one antibody, MPF was partially purified by immunoadsorption chromatography. These experiments provide the first evidence that MPF from Xenopus is a phosphoprotein that becomes thiophosphorylated upon addition of ATPγS.

Improvements in the purification of an antisteroid antiserum resistant to conventional immunoadsorption chromatography

Antibodies against dexamethasone, a synthetic steroid, have been induced in rabbits immunized with a 3-carboxymethyloxime dexamethasone derivative conjugated to bovine serum albumin. The antiserum displaying the highest affinity for dexamethasone (K D = 0.5 nM) appeared to be resistant to purification on an agarose matrix bearing the same 3-carboxy-methyloxime dexamethasone derivative. No desorption of active antibodies could be obtained whatever the eluting buffer used. Electrophoretic elution gave only poor results. However a very significant improvement in the purification of these antibodies was achieved by changing the connecting arm for steroid linkage to the agarose beads. A 17-fold purification and a 32% recovery of active specific antisteroid antibodies were obtained using a column bearing a 17β-carboxamide dexamethasone derivative. Moreover good results (23-fold purification and 30% recovery) were also obtained with a commercially available preactivated high-performance silica column derivatized with an aminated 3-carboxymethyloxime derivative of dexamethasone. In this case the more efficient diffusion of the eluting solution through the pores of a high performance stationary phase made of small diameter rigid beads probably explained the strikingly improved results, when compared to those obtained with agarose beads bearing the same dexamethasone derivative.

An antiserum against chicken hydroxyindole- O-methyltransferase reacts with the enzyme from pineal gland and retina and labels pineal modified photoreceptors

Biosynthesis of the indolic hormone melatonin has been reported in the pineal gland and retina. The terminal step of melatonin synthesis is catalysed by hydroxyindole- O-methyltransferase (HIOMT), an enzyme displaying highest levels of activity in the pineal gland and retina. Several laboratories have suggested that melatonin synthesis might take place in retinal photoreceptors and in photoreceptor-derived cells of the pineal gland. Experimental support to this hypothesis is progressively building up with the immunocytochemical identification of HIOMT-containing cells in various animal species. In the present report, HIOMT was purified from the chicken pineal gland using a one-step chromatographic procedure and an antiserum against the enzyme was obtained in the rabbit. The antiserum was further purified by immunoadsorption chromatography on chicken brain proteins. Using electroblots immunochemical labeling, HIOMT from chicken pineal gland and retina was identified as a 38-kDa protein. Pineal HIOMT was further resolved into components of different pHi-values (5.4–5.7 and 6.8), using two-dimensional gel electrophoresis. Immunoprecipitation of HIOMT activity was observed in pineal homogenates and, for the first time, in homogenates of the retina. Immunofluorescence microscopy provided the first evidence that HIOMT is contained in modified photoreceptors of the chicken pineal gland. No immunofluorescence could be observed in the retina, maybe due to the lower level of HIOMT activity in this tissue. Together, the data indicate that the antiserum may be a useful tool to study the regulation of HIOMT synthesis in the pineal gland and in the retina. Further work is required to identify HIOMT-containing cells in the retina.

Partial purification and characterization of an inactive precursor of Pseudomonas aeruginosa elastase.

An inactive precursor of the extracellular elastase of Pseudomonas aeruginosa was extensively purified by immunoadsorption chromatography of the soluble bacterial cell fraction on a column of Sepharose coupled to antielastase antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified precursor fraction revealed two major protein bands with molecular weights of about 36,000 (P36) and 20,000 (P20) that in the absence of sodium dodecyl sulfate were associated with each other. The following findings identify P36 as the elastase precursor and indicate that proteolytic processing of this molecule is required for activation: (i) P36 is larger than the elastase, and it binds antielastase antibodies; (ii) trypsin activation is associated with the disappearance of P36 and the appearance of a new protein band migrating identically with the elastase and reacting with antibodies against the elastase; (iii) peptide maps generated from P36 and the elastase are similar although not identical. P20 by itself was not recognized by antielastase antibodies. Its association with P36 accounts for its adsorption to the immunoaffinity column and suggests that it may serve in elastase secretion.

Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator.

Human recombinant single chain urokinase-type plasminogen activator (recombinant scu-PA) and a hybrid between human tissue-type plasminogen activator (t-PA) and scu-PA, obtained by ligation of cDNA fragments encoding the NH2-terminal region (amino acids 1-67) of t-PA and the COOH-terminal region (amino acids 136-411) of scu-PA, were expressed in a mammalian cell system. The proteins were purified from conditioned culture media containing 2% fetal calf serum by chromatography on zinc chelate-Sepharose, immunoadsorption chromatography on an insolubilized murine monoclonal antibody directed against urokinase, benzamidine-Sepharose chromatography, and Ultrogel AcA 44 gel filtration. Between 180 and 230 micrograms of the purified proteins were obtained per liter of conditioned medium, with a yield of approximately 18% and a purification factor of 720-1900. On sodium dodecyl sulfate gel electrophoresis under reducing conditions, the proteins migrated as single bands with approximate Mr 50,000 for recombinant scu-PA and Mr 43,000 for the t-PA/scu-PA hybrid. Following conversion to urokinase with plasmin, the proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with Km = 0.53 and 1.4 microM and k2 = 0.0034 and 0.0027 s-1, respectively. Both proteins did not bind specifically to fibrin and had a comparable degree of fibrin selectivity as measured in a system composed of a whole human 125I-fibrin-labeled plasma clot suspended in human plasma. It is concluded that this chimeric protein, consisting of the NH2-terminal "finger-like" domain of t-PA and the COOH-terminal region of scu-PA, has very similar enzymatic properties as compared to scu-PA, but has not acquired the fibrin affinity of t-PA.