Pump-1 cDNA codes for a protein with characteristics similar to those of classical collagenase family members

Pump-1 cDNA has recently been isolated by screening a human tumor cDNA library with a transin (rat stromelysin) probe under low-stringency hybridization conditions. The cDNA codes for a potential protein with significant sequence similarity to the metalloproteinases collagenase and stromelysin, but which lacks the hemopexin-like domain characteristic of these enzymes. Expression of pump-1 cDNA in cos cells using an expression vector leads to secretion of a protein of Mr 28,000 with latent, organomercurial-activatable proteinase activity. Cos cells transfected with a partial pump-1 cDNA in the vector pPROTA secrete a fusion protein between the IgG-binding domains of staphylococcal protein A and pump-1. The fusion protein binds to IgG-Sepharose, and the bound fusion protein undergoes apparent autocleavage in the presence of 4-aminophenylmercuric acetate with elution of active pump-1 species of Mr 21,000 and 19,000. Active pump-1 degrades casein, gelatins of types I, III, IV, and V, and fibronectin and can activate collagenase. Active pump-1 is inhibited by EDTA, 1,10-phenanthroline, and the tissue inhibitor of metalloproteinases. These results show that, despite the absence of a hemopexin-like domain, pump-1 is a latent secreted metalloproteinase. Postpartum rat uteri contain elevated levels of rat pump-1 mRNA. On the basis of this observation, its size, and its substrate specificity, we suggest that pump-1 might correspond to a previously described uterine metalloproteinase, matrix metalloproteinase 7.     

The complete amino acid sequence of the high molecular mass hemorrhagic protein HR1B isolated from the venom of Trimeresurus flavoviridis

Hemorrhage is a common occurrence in a victim bitten by crotalid and viperid snakes, and hemorrhagic components in these various venoms have been isolated and characterized. Previously, we have shown that a low molecular weight hemorrhagic protein (HR2a, 202 amino acid residues) isolated from the venom of Trimeresurus flavoviridis is a member of a new subfamily of metalloproteinases. We now report the complete amino acid sequence of a high molecular mass hemorrhagic protein isolated from the same venom. This protein, HR1B, is a mosaic protein composed of 416 residues containing four asparagine-linked oligosaccharide chains. The amino-terminal half (residues 1-203) of HR1B contains a metalloproteinase domain, the sequence of which is 62% identical to that of HR2a and 52% identical to that of hemorrhagic toxin d isolated from Crotalus atrox venom. The most interesting finding is that the middle region (residues 204-300) of HR1B shows a striking similarity to disintegrins, Arg-Gly-Asp-containing platelet aggregation inhibitors, recently found in several viper venoms. Interestingly, however, this region of HR1B does not contain the Arg-Gly-Asp sequence which is known to be a putative binding site in the disintegrins for the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. We also found that the carboxyl-terminal region (residues 213-336) of the middle part of HR1B shows 30% identity to residues 1543-1656 of von Willebrand factor and that the remaining region at the carboxyl-terminal end is unique and has a cysteine-rich sequence. These results suggest that the middle portion of HR1B, which shows structural similarities to the disintegrins and von Willebrand factor, may be important in synergistically stimulating hemorrhagic activity in the NH2-terminal metalloproteinase domain.     

A functionally active heavy chain derived from human high molecular weight urokinase

Human high molecular weight urokinase, a plasminogen activator, when minimally reduced with 0.01 M 2-mercaptoethanol for 10 h at pH 8.0 and 25 degrees C and then carboxymethylated with sodium iodoacetate, gave two chains, a functionally active heavy chain with about 80% of the original activity and a light chain. These two chains were found to be linked by a single interchain disulfide bond. The functionally active heavy chain can be isolated by an affinity chromatography method with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose. The light chain, which has no enzyme activity, is not adsorbed to the affinity matrix, whereas the active heavy chain was adsorbed and subsequently eluted. The active heavy chain was further purified by gel filtration on Sephadex G-100. This preparation was found to be homogeneous by both analytical and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The molecular weight of the active heavy chain was determined to be 33,000 by Sephadex G-100 gel filtration and 31,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Its specific activity, with L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide, was determined to be 208,000 IU/mg of protein. Approximately 87% active sites were found by p-nitrophenyl-p'-guanidino-benzoate titration with a molar activity of 7.41 X 10(9) IU/mmol of active site. The active heavy chain when compared to low molecular weight urokinase has a similar molecular weight, specific activity, and amino acid composition. The NH2-terminal residue found in the active heavy chain was lysine which was the same as that found in low molecular weight urokinase, whereas the NH2-terminal residues found in high molecular weight urokinase were serine and lysine. Serine is the NH2-terminal residue of the light chain of high molecular weight urokinase. The steady state kinetic parameters of activation of human Glu-plasminogen by the active heavy chain were also similar to low molecular weight urokinase, as were the amidase parameters of these enzymes. The Michaelis constants of activation (Kplg) were 2.11 and 2.21 microM, respectively; the catalytic rate constants of activation (kplg) were 51.7 and 44.1 min-1, respectively, with second order rate constants, kplg/Kplg of 24.5 and 20.2 microM-1 min-1, respectively.     

Low molecular weight plasma proteins isolated from preparations of human immunoglobulin

Low molecular weight proteins co-purified with IgG constitute 0.22% of the total protein purified from human plasma by ion-exchange chromatography on DEAE-cellulose. We have found that these low molecular weight proteins were obtained free of immunoglobulin by ultrafiltration in 5 M guanidinium chloride. Electrophoresis and isoelectric focusing in polyacrylamide gels demonstrated that this fraction of low molecular weight proteins is remarkably heterogeneous. Chromatography of an Mr 6000 to 12 000 fraction on hydroxyapatite resolved fourteen discrete protein peaks. Three of the peaks contained proteins which appeared to be homogeneous on acid-urea polyacrylamide gels. Two of these proteins were similar in composition to B2 globulin and may represent degradation products of some larger protein. The third protein was found to have an amino-terminal sequence identical to C3a. This population of low molecular weight plasma proteins has previously been shown to contain the cystic fibrosis mucociliary inhibitor and is here shown to contain two proteins similar to B2 globulin, C3a and many proteins remaining to be characterized. The presence of these low molecular weight proteins in measurable concentrations may be insufficiently appreciated in studies using 'purified' immunoglobulins as biological or chemical probes.     

Urokinase separation from cell culture broth of a human kidney cell line

A single step ion-exchange chromatography on a sulfo-propyl (SP)- Sepharose column was performed to separate both the high molecular weight (HMW)- and low molecular weight (LMW)- forms of enzymatically active urokinase type plasminogen activator from human kidney (HT1080) cell culture media. The level of urokinase secreted by the cell line reached to about 145 Plough units/ml culture broth within 48 h of cultivation. The conditioned cell culture media was applied directly to the column without any prior concentration steps. Polyacrylamide gel electrophoresis of the column eluates in the presence of sodium dodecyl sulphate showed that the cell line secretes three forms of two-chain high molecular weight (HMW) urokinase of molecular weights (M(r)) 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW) forms of M(r) 22,000 and 20,000; proteolytic cleavage products of HMW, were also found. The HMW and LMW forms had intrinsic plasminogen dependent proteolytic activity as judged by zymographic analysis. The specific activity of the pooled peak fractions increased (approximately 93-fold) to values as high as 1481 Plough units/ mg protein. Both HMW as well as LMW forms were obtained in significantly high yields.     

Extended reading frame of a ubiquitin gene encodes a stable, conserved, basic protein

Antibodies specific for the 80-amino acid hypothetical protein encoded by the in-frame, 3'-extension of a human ubiquitin gene were produced in rabbits by immunization with a 14-residue synthetic peptide. When used to probe HeLa cell extracts for the non-ubiquitin product of this natural fusion gene, the antipeptide sera detected a protein with an apparent molecular weight of 16,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An immunoreactive protein of identical mobility was detected in organisms ranging from Acanthamoeba to man, indicating that the extension protein, like ubiquitin, is highly conserved. The immunoreactive protein was isolated from calf thymus, and direct sequencing revealed the first 16 amino acids to be identical to those predicted from the extension portion of the human cDNA. Thus, ubiquitin was no longer present at the amino terminus. The purified bovine extension protein failed to react with a ubiquitin-specific antibody indicating the absence of isopeptide-linked ubiquitin as well. Moreover, by denaturing gel permeation chromatography the extension has a molecular weight of 10,000 Da, a value that corresponds more closely to the size of the extension alone (9,000 Da) than to the intact fusion protein (17,500 Da). The extension protein, which was found in both cytoplasmic and nuclear fractions of HeLa cells, persisted at high levels when protein synthesis was blocked with cycloheximide or puromycin. These results show that the 80-residue extension protein is the stable, processed product of the ubiquitin fusion gene.     

A proenzyme form of human urokinase

A culture of the human epidermoid carcinoma HEp 3 produces a plasminogen activator of Mr = 53,000 which we have purified to apparent homogeneity from serum-free conditioned medium by the combination of immunoaffinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The highly purified protein has the following properties: 1) It is indistinguishable from urinary urokinase in electrophoretic mobility, in immunodiffusion, and in autoradiographically visualized tryptic peptide maps obtained from the 125I-labeled proteins. 2) The HEp 3 protein differs from urinary urokinase in the following respects: (a) although the apparent molecular weights of the two are identical (Mr = 53,000), the urinary enzyme consists of two polypeptide chains, whereas the HEp 3 protein is a single chain form. (b) Urinary urokinase can be labeled easily by incubation with radioactive diisopropylfluorophosphate but the HEp 3 protein cannot. (c) When assayed by the hydrolysis of a synthetic chromogenic peptide substrate, the HEp 3 enzyme has less than 1% of the catalytic activity of urinary urokinase. 3) On controlled exposure to plasmin, the HEp 3 protein is converted to an active enzyme that is identical with urinary urokinase in molecular weight, polypeptide chain composition, diisopropylfluorophosphate labeling, and specific catalytic activity. We conclude that the HEp 3 protein is a proenzyme that can be converted to active two-chain urokinase by plasmin, probably by a single proteolytic nick in the polypeptide chain.     

Low molecular weight serine proteinase inhibitors of the human intervertebral disc

Human lumbar disc tissue when extracted with 4M GuHCl and subjected to dissociative CsCl density gradient ultracentrifugation yielded trypsin inhibitor activity in the low bouyant density fractions (rho less than or equal to 1.38 g/ml). Disc proteoglycans sedimented in the high bouyant density fractions (rho greater than or equal to 1.5 g/ml). Sephadex G75F gel filtration of the low bouyant density protein fractions afforded a major low molecular weight (Kav = 0.5) trypsin inhibitor pool which was further purified by trypsin affinity chromatography. This latter step facilitated separation of the trypsin inhibitors from neutral proteinase activity also present. The trypsin inhibitor fraction so isolated was shown to possess potent inhibitory activity against a range of human serine proteinases including leukocyte elastase and cathepsin G, urokinase, kallikrein, plasmin and thrombin. Significantly this serine proteinase inhibitor preparation effectively prevented degradation of proteoglycans by a neutral proteinase also isolated from the human intervertebral disc.     

Protein L: an immunoglobulin light chain-binding bacterial protein. Characterization of binding and physicochemical properties

Protein L, a cell wall molecule of the bacterial species Peptostreptococcus magnus with affinity for immunoglobulin (Ig) light chains, was isolated after solubilization of the bacterial cell walls with mutanolysin or from the culture medium by a single affinity chromatography step on human IgG-Sepharose. A major protein band with an apparent molecular weight of 95,000 was obtained from both sources. The protein from the growth medium was size heterogeneous. From 1 ml of packed bacteria was prepared 0.92 mg of the mutanolysin-solubilized protein L (73% yield), whereas 4.1 mg of spontaneously released protein L (49% yield) was purified from the corresponding culture medium. The Mr of protein L was estimated to 76,000 by gel chromatography in 6 M guanidine HCl. Using this Mr value, the Stokes radius and the frictional ratio of protein L were determined to 4.74 nm and 1.70, respectively, suggesting an elongated fibrous molecule. No disulfide bond or subunit structure could be shown. The amino-terminal amino acid sequences of the whole protein and two internal non-IgG-binding tryptic fragments were determined and found to be unique. One of the tryptic fragments showed homology (40% identical residues) to a sequence within the cell wall-binding region of protein G, the Fc-binding protein of group C and G streptococci. The binding specificity of protein L was directed to the light chains of immunoglobulins; the affinity constant for polyacrylamide-coupled kappa-chains was 1.5 x 10(9) M-1 and for IgG, IgA, and IgM around 1 x 10(10) M-1. Maximal binding was achieved between pH 7 and 10. The binding to lambda-chains was too weak for determination of the affinity constant. 125I-Protein L was also shown to bind to mouse immunoglobulins. It could be used for detection of antigen-bound polyclonal and monoclonal antibodies in Western blots. This shows that the protein L/light chain reaction was not obstructed by occupation of the antigen-binding site. Finally, protein L and kappa-chains of human Ig formed precipitates upon double immunodiffusion analysis, an indication of at least two binding sites on protein L.     

Purification and characterization of a novel low molecular weight form of single-chain urokinase-type plasminogen activator

A low Mr form (Mr 32,000) of single-chain urokinase-type plasminogen activator (scu-PA) was isolated from conditioned culture medium of a human lung adenocarcinoma cell line, CALU-3 (ATCC, HTB-55). The purified material (scu-PA-32k) consists of a single polypeptide chain and is immunologically similar to Mr 33,000 urokinase. Its NH2-terminal sequence is identical to that beginning at Leu-144 of Mr 54,000 urokinase. Whereas low Mr urokinase is derived from mature Mr 54,000 scu-PA by limited hydrolysis by plasmin first of the Lys-158-Ile-159 peptide bond and then of the Lys-136-Lys-137, scu-PA-32k is generated by specific hydrolysis of the Glu-143-Leu-144 peptide bond by an unidentified protease. scu-PA-32k resembles its Mr 54,000 scu-PA counterpart by its very low activity on chromogenic substrates for urokinase, by plasminogen-dependent fibrinolytic activity on fibrin plates, and by the lack of specific binding to fibrin. It activates plasminogen directly with high affinity, Km = 0.9 microM, but low turnover number, kcat = 0.0028 s-1. It is converted to fully active two-chain urokinase by plasmin with Km = 12 microM and kcat = 0.3 s-1. Like Mr 54,000 scu-PA, it causes significant lysis of a 125I-labeled fibrin clot in human plasma with relatively less fibrinogen breakdown as compared to urokinase. scu-PA-32k, which also has conserved fibrin specificity, represents a molecular variant which may be more suitable for large scale production as a fibrin-specific thrombolytic agent by recombinant DNA technology.     

Isolation and characteristics of urokinase-type plasminogen activator from a culture of human embryo lung fibroblasts

Plasminogen activator from conditioned medium of human embryonal lung fibroblasts was purified by phosphocellulose P11 chromatography, followed by p-aminobenzamidine-agarose chromatography. Two forms of plasminogen activators were separated by chromatography on the heparin-sepharose. The high molecular weight form (53 kDa) with specific activity 130 000 IU/mg consists of two polypeptide chains (31 kDa and 20 kDa) and exhibits strong affinity for fibrin-celite, lysine-sepharose and heparin-sepharose. The low molecular weight form (32 kDa, 190 000 IU/mg) also binds to these sorbents, but more weakly, and its properties are very similar to those of low molecular weight urokinase. Activity of both forms of plasminogen activators are inhibited by monoclonal antibodies against urokinase. A number of enzymological chromatographic and immunological properties indicates, that the plasminogen activator from lung fibroblasts is of urokinase type.     

The reactive site of human inter-alpha-trypsin inhibitor is in the amino-terminal half of the protein

Human inter-alpha-trypsin inhibitor has been found to inactivate human trypsin, chymotrypsin, neutrophil elastase and cathepsin G. The protein was cleaved into two major fragments without loss of activity by incubation with Serratia marcescens metalloproteinase, and these were separated by ion-exchange chromatography. Inhibitory activity was found in only one of the fragments, the amino-terminal sequence of which was found to be identical with that of the native protein, as well as with that reported earlier for the urinary trypsin inhibitor. It may thus be concluded that the reactive site of the inter-alpha-trypsin inhibitor is located in the amino-terminal region.     

Renal gamma-glutamyl transpeptidases: structural and immunological studies

Mammalian kidney gamma-glutamyl transpeptidases are compared with respect to subunit size, amino-terminal sequences of the two subunits, immunological, and some catalytic properties. The species-related variation in the apparent molecular weight of the subunits has been shown to be primarily due to the extent and nature of protein glycosylation. Using antibodies raised against the native enzymes and isolated sodium dodecyl sulfate-treated subunits, it is shown that the transpeptidases share some antigenic determinants. Some of these determinants in the highly glycosylated transpeptidase subunits can be detected by the antibodies only upon deglycosylation of the subunits. The amino-terminal sequences of the subunits exhibit considerable homology, in agreement with the immunological data. Thus, there are two segments of identity (3 and 5 residues in length, respectively) in the first 17 amino-terminal residues of the heavy subunits of rat, bovine, dog, and human kidney transpeptidases (papain-solubilized). Of particular interest is the finding of 91 to 96% identity in the first 23 amino-terminal residues of the small subunit of these transpeptidases. The small subunit contains the gamma-glutamyl binding site of the enzyme. There are three segments of identity (7, 6, and 8 residues in length, respectively) in the first 23 residues, each separated by either a Ser or an Ala residue. The first 7 amino-terminal residues of the small subunit in all four species are identical, indicating a high degree of specificity in the proteolytic processing of the common, single-chain precursor of the two subunits. Differences noted between transpeptidases in their relative acceptor specificity and in their susceptibility to inactivation by the glutamine antagonist, AT-125 (acivicin), must reflect subtle structural differences in their active center domains.     

Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase.

The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.     

Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator.

A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred.     

A new form of amyloid protein associated with chronic hemodialysis was identified as beta 2-microglobulin

Amyloid fibrils were isolated from amyloid-laden tissue obtained from a chronic hemodialysis patient with carpal tunnel syndrome. After solubilization in guanidine HCl, a significant amount of the protein was located in a homogeneous low molecular weight fraction. The protein was found to be identical to beta 2-microglobulin, with regard to its molecular weight of 11,000, amino acid composition and 16 amino-terminal amino acids: Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-Arg-His-Pro-Ala-Glu-. These results demonstrate that the amyloid associated with chronic hemodialysis contains as major component a new form of amyloid fibril protein that is homologous to beta 2-microglobulin.     

Immunochemical characterization of two activator proteins stimulating enzymic sphingomyelin degradation in vitro. Absence of one of them in a human Gaucher disease variant

Two nonenzymic activator proteins shown previously to strongly stimulate enzymic sphingomyelin degradation in vitro were purified from human Gaucher type 1 and control spleen. Activator A1 (molecular mass 6,500 Da) had affinity for ConA-Sepharose, while activator A2 (molecular mass 3,500 Da) did not. Monospecific antibodies to each activator protein were prepared in rabbits by immunization with protein purified from type 1 Gaucher spleen. A1 and A2 activators from Gaucher type 1 spleen were shown to be immunochemically identical to A1 and A2 activators from control spleen. However, A1 and A2 activators, whether isolated from Gaucher type 1 or control spleen, were shown to be distinct proteins. Immunochemical examination of all collected fractions during the purification revealed the existence of a third activator (molecular mass 6,000 Da), which was antigenically identical to A1 activator but had no affinity for ConA-Sepharose. The two forms of A1 activator showed similar mobility on immunoelectrophoresis differing from that of A2 activator. Fibroblast extracts from controls and patients with different variants of Gaucher disease were investigated using immunodiffusion against antisera to A1 or A2 activator. In contrast to normal and Gaucher (types 1, 2 and 3) cell extracts, those of a Gaucher patient with normal glucosylceramidase activity had no visible precipitin line towards the antiserum against the two forms of A1 activator. The lack of crossreacting material to antibodies against A1 activator was confirmed by radial immunodiffusion and rocket immunoelectrophoresis. A1 activator stimulated the basal glucosylceramidase activity 5-6 fold in fibroblasts from this patient, whereas the normal effect was only a 1.2-1.5-fold stimulation. The immunological results together with the biochemical data provide evidence for the lack of an activator protein in a variant form of human Gaucher disease for the first time.     

Covalent molecular weight approximately 92 000 hybrid plasminogen activator derived from human plasmin amino-terminal and urokinase carboxyl-terminal domains

The preparation of a new class of covalent hybrid plasminogen activators containing the fibrin-binding domains of human plasmin(ogen) and the catalytic active center of human urokinase will be described. Hybridization of the sulfhydryl form of the NH2-terminal plasmin-derived heavy (A) chain (PlnA) with the sulfhydryl form of the COOH-terminal urokinase-derived active heavy (B) chain (u-PAB) was carried out; a covalent PlnA-u-PAB hybrid plasminogen activator was prepared. The sulfhydryl form of PlnA (PlnA(SH)2) was isolated from reduced Lys-2-plasmin by L-lysine-substituted Sepharose column chromatography. For the isolation of the sulfhydryl form of u-PAB (u-PAB(SH], high molecular weight urokinase was adsorbed onto a benzamidine-Sepharose column and reduced with 100 mM 2-mercaptoethanol on the column. The urokinase NH2-terminal light (A) chain was washed off the column, and the u-PAB(SH) chain was eluted from the column. The specific activity of the isolated u-PAB(SH) chain was determined to be 242 000 IU/mg of protein. The PlnA(SH)2 and u-PAB(SH) chains were mixed at a molar ratio of PlnA(SH)2 to u-PAB(SH) of 3:2; the reducing agents were then removed by gel filtration. The hybridization (reoxidation) reaction was allowed to proceed for 48 h at 4 degrees C. The covalent hybrid activator, in 40% yield, was purified from the reaction mixture to homogeneity, by a sequential affinity chromatography method with L-lysine-substituted Sepharose followed by anti-low molecular weight urokinase IgG-Sepharose, and then gel filtration through Sephadex G-150.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and partial characterization of a tubulin-like protein from human and swine synaptosomal membranes.

Synaptic membranes from human and swine brains were solubilized with 8 M urea and the proteins were reduced and alkylated. A protein was isolated from both sources and had identical amino acid compositions and molecular weights as determined by electrophoresis on polyacrylamide-sodium dodecylsulfate gels and by ion-exchange chromatography and gel filtration on Bioglas 1000. The apparent molecular weight of the protein was 53 000 on the acrylamide-sodium dodecylsulfate gels. Neither neutral sugars nor sialic acid was a significant component of the protein. When the proteins were digested with trypsin and the resultant peptides subjected to chromatography (n-butanol/acetic acid/water) and electrophoresis (pH 3.7) the peptide maps were identical. The protein comprises 1-2 percent of the total synaptosomal protein. With regard to amino acid composition, molecular weight, peptide map characteristics, behavior on DEAE-cellulose columns, electrophoretic mobility and sugar content, the synaptic protein is quite similar to the monomer of swine tubulin.