Purification of human factor VIII:C and its characterization by Western blotting using monoclonal antibodies

Human factor VIII:C has been purified over 300 000-fold from cryoprecipitate by polyelectrolyte purification followed by affinity chromatography on Sepharose linked to antibody to factor VIIIR:Ag (monoclonal or polyclonal) and Sepharose linked to monoclonal antibody to factor VIII:C. The purified material has been analyzed by polyacrylamide gel electrophoresis (PAGE) and Western blotting using monoclonal antibodies. PAGE shows predominant bands at 360K (unreduced), 210K, and 90K and an 80K/79K doublet; Western blotting showed all the monoclonal antibodies used bound the 360K form. In a small-scale purification, plasma from blood taken directly into thrombin inhibitor Kabi S-2581 was applied directly to the monoclonal anti-factor VIII:C column. Western blot analysis of this material showed the 360K band on reduction. The purified factor VIII:C could be activated 13-fold by human thrombin. Gel analysis of the activated material showed intensification followed by fading of the band at 90K and generation of bands at 70K/69K, 55K, and 40K. Western blotting shows that the 70K/69K doublet derives from the 80K/79K moiety and the 40K peptide derives from the 90K and is presumed to contain the active site. From these studies an epitope map of the factor VIII:C molecule has been constructed.     

Isolation and characterization of proteins that stimulate the activity of mammalian DNA methyltransferase

Previously, the purification of DNA methyltransferase from murine P815 mastocytoma cells by immunoaffinity chromatography was described (Pfeifer, G.P., Grünwald, S., Palitti, F., Kaul, S., Boehm, T.L.J., Hirth, H.P. and Drahovsky, D. (1985) J. Biol. Chem. 260, 13787-13793). Proteins that stimulate the enzymatic activity of DNA methyltransferase have been purified from the same cells. These proteins, which partially coelute with DNA methyltransferase from DEAE-cellulose and heparin-agarose, are separated from the enzyme during the immunoaffinity purification step. A further purification of the stimulating proteins was achieved by butanol extraction, DEAE-cellulose chromatography and gel filtration on Superose 12. Two DNA methyltransferase-stimulating protein fractions were obtained. SDS-polyacrylamide gel electrophoresis of one fraction showed a single polypeptide with a molecular mass of 29 kDa. The second fraction consisted of 5 or 6 polypeptides with molecular masses 78-82 and 51-54 kDa. The proteins stimulate both de novo and maintenance activity of DNA methyltransferase about 3-fold. They enhance the methylation of any natural DNA and of poly[(dI-dC).(dI-dC)] but inhibit the methylation of poly[(dG-dC).(dG-dC)]. The purified proteins do not form a tight complex with DNA methyltransferase; however, they bind both to double-stranded and single-stranded DNA. The sequence specificity of DNA methyltransferase is obviously altered in presence of these proteins.     

Acetolactate synthase from Brassica napus: Immunological characterization and quaternary structure of the native enzyme

Acetolactate synthase (ALS, AHAS; EC 4. 1. 3. 18) from Brassica napus has been partially purified and characterized using polyclonal antibodies. Following denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blot analysis, 65 and 66 kDa ALS subunit polypeptides were immunologically detected, along with a novel 36 kDa polypeptide which cross-reacted with the anti-ALS antibody. Partial peptide sequencing of the 36 kDa peptide revealed significant similarity to plant aldolase proteins. ALS activity from stromal extracts fractionated by gel filtration chromatography as a single species of estimated molecular mass of 124 kDa, while comparative sedimentation coefficient in glycerol gradients indicated a corresponding molecular mass of 132 kDa. The results suggest that the native enzyme is a dimer of 65 and/or 66 kDa subunits. Anion exchange chromatography resolved two classes of ALS activity of equal native molecular weight, but which exhibited different properties with respect to subunit structure, sensitivity to inhibition by chlorsulfuron and feedback inhibition by branched chain amino acids.     

Human eosinophil cytotoxicity-enhancing factor. Purification, physical characteristics, and partial amino acid sequence of an active polypeptide

Medium conditioned by PMA/LPS-stimulated U937 cells was processed for the purification of an eosinophil cytotoxicity-enhancing factor (ECEF) by the following sequence: 1) phenyl-Sepharose chromatography; 2) DEAE-cartridge chromatography; 3) preparative SDS-gel electrophoresis; and 4) reversed-phase HPLC. This resulted in the isolation of a 10 kDa polypeptide with ECEF activity. Purified material from 21 different preparations enhanced eosinophil killing of antibody-coated Schistosoma mansoni schistosomula by a mean of 206% (increase from 13.2 +/- 7.9% to 40.4 +/- 20.2% of targets killed, p less than 0.0001). Activity was maximal at a concentration of 20 ng ECEF polypeptide/ml and half-maximal between 0.8 and 4 ng/ml. Antibody specific for the 10 kDa polypeptide precipitated ECEF activity from a crude preparation and, by Western blot analysis, reacted only with a 10 kDa species in that preparation. The following N-terminal amino acid sequence was determined for the purified polypeptide: Val-Lys-Gln-Ile-Glu-Ser-Lys-Thr-Ala-Phe-Gln-Lys-Ala-Leu- -Ala- Gly- -Lys-Leu....Computer search showed that this sequence is unrelated to other known protein sequences. Thus, the ECEF polypeptide is a newly defined monokine, with the ability to enhance eosinophil cytotoxic function in vitro. This monokine may be an important regulator of eosinophil function in inflammation in vivo.     

Purification and characterization of single-chain urokinase-type plasminogen activator from human cell cultures

A urokinase-type plasminogen activator was purified from conditioned media of several human cell cultures, but preferably from the human lung adenocarcinoma line CALU-3 (ATCC, HTB-55), using a combination of chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, and Sephadex G-100. Final yields of 65-100 micrograms/liter of starting material were obtained with a 290-fold purification factor and a recovery of 30%. The purified plasminogen activator consists of a single polypeptide chain with Mr 54,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is very similar or identical to single-chain urokinase-type plasminogen activator on the basis of immunodiffusion, amino acid composition, and the lack of specific binding to fibrin. It has very low amidolytic activity on Pyroglu-Gly-Arg-rho-nitroanilide and is converted to two-chain urokinase by limited exposure to plasmin. It has a specific activity of 60,000 IU/mg on fibrin plates and directly activates plasminogen following Michaelis-Menten kinetics with Km = 1.1 microM and kappa cat = 0.0026 S-1. It is concluded that the plasminogen activator purified from CALU-3-conditioned media is physically and kinetically identical to single-chain urokinase-type plasminogen activator. With the present straightforward purification method and a readily available source, sufficient amounts of single-chain urokinase-type plasminogen activator can be obtained for more detailed investigations of its biochemical, biological, and thrombolytic properties.     

Purification and characterization of UDP-N-acetyl-D-glucosamine:dolichol phosphate N-acetyl-D-glucosamine-1-phosphate transferase involved in the biosynthesis of asparagine-linked glycoproteins in the mammary gland

The GlcNAc-1-P-transferase that initiates the dolichol cycle for the biosynthesis of asparagine-linked glycoproteins has been purified from the lactating bovine mammary gland. After solubilization from microsomes with 0.25% Nonidet P-40, the enzyme activity was stabilized with 20% glycerol, 20 micrograms/ml phosphatidylglycerol, 5 microM dolichol phosphate, and 2.5 microM UDP-GlcNAc. The purification protocol involved (NH4)2SO4 precipitation, gel filtration on Sephacryl S-300, DEAE-TSK, and hydroxylapatite chromatography. The purified enzyme was devoid of several readily detectable glycosyltransferases of the dolichol cycle. It showed two bands (A, 50 kDa and B, 46 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after either Coomassie Blue or silver staining. Antisera (anti-A and anti-B) raised against individual bands A and B inhibited the enzyme activity in solubilized microsomes. Each of the partially purified antibodies recognizes both bands A and B on Western blots of the enzyme; with the solubilized microsomes, the antibodies also recognize an additional polypeptide of approximately 70 kDa. When radioiodinated microsomes were immunoprecipitated with anti-B and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, again bands of 46, 50, and 70 kDa were observed. The peptide mapping of 50 and 46 kDa bands of the purified enzyme by chemical cleavage with N-chlorosuccinimide gave similar fragmentation patterns. The results indicate that either 70 kDa band is a precursor form of the enzyme or this polypeptide, representing the native enzyme or its subunit, is proteolyzed to smaller, enzymatically active peptide(s) of 50 and 46 kDa during purification despite the inclusion of several inhibitors against serine-proteases in all buffers used for tissue homogenization and enzyme purification. A number of properties of the purified enzyme, including its specific activation by Man-P-Dol were also characterized.     

Isolation and characteristics of endonuclease tightly bound to alpha-polymerase from the rat liver

Three major polypeptides are found in purified DNA polymerase alpha from rat liver: 160, 77 and 58 kDa. The electrophoretic analysis has identified polypeptide 160 kDa as the catalytically active subunit of DNA polymerase alpha. The other two polypeptides showed no DNA polymerase activity. Individual polypeptide p77 kDa purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to produce antibodies in rabbits. Immunoblot analysis indicated that the complex DNA polymerase alpha-3'-5'-exonuclease contained polypeptide p77 kDa. To elucidate the function of the p77 kDa protein we have prepared an immunoabsorbent column with antibodies against the p77 kDa polypeptide. The antibody column purified p77 kDa protein was homogeneous according to sodium dodecyl sulfate gel electrophoresis. The activity of alpha-polymerase was increased approximately 10-fold as a result of purification of DNA polymerase alpha from the p77 kDa protein. The in vitro experiments showed the identity of the p77 kDa polypeptide to endonuclease. It cleaved both single-stranded and double-stranded DNA. The function of endonuclease p77 kDA in complex with DNA polymerase alpha remains obscure.     

Isolation and functional reconstitution of the aspartate/glutamate carrier from mitochondria

The aspartate/glutamate carrier from beef heart mitochondria was solubilized by the detergent dodecyloctaoxyethylene ether (C12E8) in the presence of high concentrations of ammonium acetate. After separating the bulk amount of contaminating proteins by differential solubilization and by hydroxyapatite centrifugation chromatography, the aspartate/glutamate carrier was purified by high-performance liquid chromatography on hydroxyapatite. During the purification process, the aspartate/glutamate carrier as well as other transport proteins was identified by functional reconstitution. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis the purified aspartate/glutamate carrier protein appears as a protein band with an apparent molecular mass of 68 kDa. Small amounts of some contaminating proteins mainly at 31 kDa were also found. Since the ADP/ATP carrier has an apparent molecular mass of 31 kDa in SDS-gel electrophoresis, possible contamination by the nucleotide carrier was analyzed by immunological methods. The enrichment of the aspartate/glutamate carrier--based on functional reconstitution--was about 570-fold, the protein yield was 0.1%.     

High-yield purification of a pp60c-src related protein-tyrosine kinase from human platelets

A protein-tyrosine kinase (PTK, EC 2.7.1.112) from human platelets was purified with high yield. Purification of the enzyme involved sequential chromatography on casein-agarose, tyrosine-agarose, heparin-Sepharose and hydroxylapatite. The procedure resulted in substantially enriched 54/52 kDa polypeptides on SDS-polyacrylamide gel electrophoresis and a yield of about 25% in PTK activity. About 250 micrograms of purified protein could be obtained from 1 g of cell protein. The purification factor varied between 1000 and 1500. Determination of the molecular mass of the purified PTK under nondenaturating conditions by molecular sieve chromatography revealed that the enzyme is a monomer of about 50 kDa. Among various protein substrates tested, casein was most prominently phosphorylated. All substrates were exclusively phosphorylated at tyrosine residues. Autophosphorylation at tyrosine residues of the 54/52 kDa proteins was observed in the presence of Mg2+ or Mn2+. At each purification step, the 54/52 kDa proteins were precipitated by sera from tumor-bearing rabbits immunoprecipitating pp60src, but not by control sera. The amount of the immunoprecipitated purified 54/52 kDa phosphoproteins was directly proportional to the amount of antiserum used. Partial peptide mapping by V8 proteinase showed a 26 kDa tyrosine-phosphorylated fragment for the 54 and the 52 kDa proteins as well as for the pp60c-src molecules of intact platelets. All these data indicated that purified PTK is closely related to pp60c-src of human platelets. Using casein as a substrate for the purified enzyme, the Km for ATP was 4 microM and the Vmax for the reaction was 2.0 nmol/min per mg.     

Purification and Partial Characterization of an Antimicrobial Peptide Produced by a Novel <Emphasis Type="Italic">Bacillus</Emphasis> sp. Isolated from the Amazon Basin

An antimicrobial peptide produced by a new Bacillus species isolated from the Amazon Basin was purified and characterized. The antimicrobial peptide was purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography, and after the final purification step, one active fraction was obtained, designated BLS P34. Direct activity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was observed. A single band on SDS-PAGE suggested that the peptide was purified to homogeneity and had a molecular mass of about 5 kDa. The molecular weight (MW) was accurately determined by mass spectroscopy as 1456 Da. The purified BLS P34 remained active over a wide temperature range and was susceptible to all proteases tested.     

Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands

CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, CMP-neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP-neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography. The protein was homogeneous on SDS-PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa). The identity of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I-ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP-neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.     

Purification and characterization of a high-affinity binding protein for pancreatic-type phospholipase A2.

A high-affinity and specific binding site for mammalian group I phospholipase A2 (PLA2-I) was found on the membranes of bovine corpus luteum. Affinity labeling experiments revealed that PLA2-I binds to a single polypeptide with a mass of 190-200 kDa. The PLA2-I binding protein in the membranes was solubilized in an active form with n-octyl beta-D-thioglucoside, and then purified approx. 16,000-fold. The purification procedures consisted of diethylaminoethyl-Sephacel chromatography, PLA2-I-affinity gel chromatography and gel-filtration high-performance liquid chromatography on a TSKgel G3,000SWXL column. The final preparation migrated as a single molecular species of 190 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and identification of the 190 kDa protein as the PLA2-I binding protein was demonstrated by ligand blotting analysis. The purified protein possessed a binding capacity with high affinity and specificity for a mammalian mature type of PLA2-I. Treatment of the purified material with N-glycosidase F resulted in increased mobility of the protein on SDS-PAGE as well as considerable abolition of the PLA2-I binding activity, thus suggesting the requirement of the carbohydrate moiety of the PLA2-I binding protein for receptor-ligand interactions.     

A Retinal Ganglion Cell Neurotrophic Factor Purified from the Superior Colliculus

Dissociated neonatal rat retinal ganglion cells can be maintained by the addition of an extract from the neonatal superior colliculus. This extract can support 95% of ganglion cells over 24 h in culture; in addition it promotes the expression of neurites from these cells. This report describes the purification of a neurotrophic factor from the superior colliculus which supports the survival of 80% of retinal ganglion cells over 24 h in vitro. The purification procedure involves a combination of dye-ligand, anion-exchange, and molecular sieve chromatography. The purified neurotrophic factor has a Stokes radius of approximately 200 A using molecular sieve chromatography in the presence of a chaotropic agent. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified factor indicates that it is a glycoprotein that migrates with a molecular mass greater than 400 kDa. Further characterization of this high-molecular-mass glycoprotein by enzymatic digestion demonstrated that it is a chondroitin sulfate proteoglycan. This factor is clearly distinguishable from other neurotrophic factors that have an effect on retinal ganglion cells such as brain-derived neurotrophic factor and fibroblast growth factor. The chondroitin sulfate proteoglycan from the neonatal superior colliculus is the first proteoglycan to be identified as a neurotrophic factor.     

Production of multiple forms during purification of Streptomyces aureofaciens DNA polymerase: a study using nondenaturing polyacrylamide gradient gel electrophoresis.

A modified method for the detection of DNA polymerases in cell extracts and purified enzyme preparations after electrophoresis in polyacrylamide gradient cylindrical gels is described. The technique, which is based on direct assay of activity in a reaction mixture during elution of DNA polymerases from gel slices, was applied to the pursuit of enzyme forms of Streptomyces aureofaciens DNA polymerase during purification procedure. In a crude extract of S. aureofaciens mycelium many catalytically active forms of DNA polymerase ranging from 35 to 860 kDa were detected. After purification, that included mycelium homogenization, precipitation of nucleic acids by polyethylene glycol, DEAE-Sephadex, QAE-Sephadex and DNA-Sepharose chromatography, higher molecular mass forms of more than 172 kDa have not been found. The lower molecular mass forms were separated into two groups by DNA-Sepharose chromatography. On the basis of their characterization, it is assumed that the lower molecular mass forms are produced by proteolysis and the major form found after purification of S. aureofaciens DNA polymerase in the presence of suitable proteinase inhibitors should be a protein of 172 kDa.     

Structural characterization of human interferon gamma. Heterogeneity of the carboxyl terminus

Natural human interferon gamma(IFN-gamma) was purified from the conditioned medium of peripheral blood leukocytes using selective silica gel adsorption and antibody-affinity chromatography. SDS-PAGE and Western blot analysis demonstrated three major species with molecular masses of 25 kDa, 20 kDa and 17 kDa. Structural analysis of this natural IFN-gamma preparation demonstrated a pyroglutamate residue at the amino terminus and a heterogeneous carboxyl terminus. The longest and most predominant polypeptide was 138 amino acids in length, which is five residues shorter than the sequence predicted from the cDNA. The presence of multiple-carboxyl-terminal forms indicated possible proteolytic processing during induction or protein purification. Limited proteolytic digestion of full-length recombinant IFN-gamma with endoproteinase Lys-C and trypsin revealed that the carboxyl-terminal 15 residues could be released under conditions in which the core portion of the polypeptide chain remained intact. Thus, the heterogeneity of natural IFN-gamma may be explained by partial proteolytic degradation of the molecule and differences in the degree of glycosylation as previously reported [Rinderknecht, E., O'Conner, B. H. & Rodriguez, H. (1984) J. Biol. Chem. 259, 6790-6797].     

Purification and some characteristics of the human coagulation factor VII.

1. A purification procedure for factor VII (proconvertin) from human plasma is described. The procedure involves barium sulphate adsorption and elution. DEAE-Sephadex column chromatography, barium sulphate adsorption and elution, heparin-Sepharose column chromatography, preparative disc gel electrophoresis and finally adsorption with antiserum to prothrombin coupled to Sepharose and antiserum to albumin coupled to Sepharose. This procedure gave an approximately 8 . 10(5)-fold purification. 2. The factor VII obtained from the electrophoresis step was mainly a single-chain protein with an apparent molecular weight of 53000 +/- 2000. 3. After the final purification step, additional forms of factor VII, resulting from a fragmentation of the factor VII molecule were detected. 4. Amino acid composition data of the purified factor VII are given. 5. Antisera were raised in two different rabbits by injection of the purified factor VII. The antisera obtained gave a good titre against the factor VII activity and were not directed against any of the three other vitamin-K-dependent coagulation factors.     

Purification of factor VIII:c coagulant activity from rat liver nonparenchymal cell culture medium by immobilized metal ion affinity chromatography

The purification of factor VIII:c coagulant activity on the basis of its affinity for calcium is described. For this purpose, use was made of a recently introduced chelating matrix, i.e., carboxymethylated aspartic acid agarose, coupled with calcium--thereby creating a gel with specificity comparable with biospecific affinity chromatography. In a single step factor VIII:c activity was purified from rat liver nonparenchymal cell culture medium with a purification factor of 85-fold. The material exhibits a single band on polyacrylamide gel electrophoresis.     

Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.)

Porphobilinogen deaminase catalyzes the condensation of four porphobilinogen monopyrrole units into hydroxymethylbilane, a linear tetrapyrrole necessary for the formation of chlorophyll and heme in higher plant cells. We report the purification to homogeneity of a chloroplast-localized form of the enzyme from pea (Pisum sativum L.) by a novel purification scheme involving dye-ligand affinity chromatography. The purified chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by size-exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the protein is acidic. The activity of the enzyme shows different levels of sensitivity to divalent cations and is most sensitive to FE2+. The amino terminus of pea enzyme has been obtained by microsequencing and determined to bear little similarity to the amino acid sequences of porphobilinogen deaminases purified from other organisms. Polyclonal antisera elicited against the purified protein has been used to examine the abundance and cellular distribution of the enzyme.     

The effect of carbohydrate depletion on procoagulant activity and in vivo survival of highly purified human factor VIII

Human factor VIII procoagulant protein (factor VIII) was purified using a modification of our previously described method, in which Sephacryl S-400 elution, rather than QAE-cellulose chromatography, served as the final purification step. The protein had a specific activity of more than 2500 U/mg and consisted of a single polypeptide (Mr 100 000) when analyzed by SDS-polyacrylamide gel electrophoresis. Factor VIII was shown to be a glycoprotein by staining with periodic acid-Schiff's reagent following electrophoresis. Treatment of factor VIII with a mixture of exo- and endoglycosidases caused a reduction by about 50% in the intensity of periodic acid-Schiff staining, as determined by scanning densitometry, and an increase in electrophoretic mobility (equivalent to a new Mr 95 000). Removal of this portion of the total carbohydrate had no significant effect on factor VIII clotting activity or on thrombin potentiation of clotting activity. The in vivo survival curves of a native and sugar-depleted 125I-labeled factor VIII both showed similar patterns of initial rapid decay to 60 and 40% activity, respectively, followed by a one-half decay time of 4 h for both. These results suggest that the carbohydrate portion of human factor VIII does not contribute significantly to either clotting function in vitro or to biological turnover in vivo.     

Alpha-transforming growth factor secreted by untransformed bovine anterior pituitary cells in culture. I. Purification from conditioned medium

A 6-kDa alpha-transforming growth factor (TGF) was purified 100,000-fold to homogeneity from the culture fluid conditioned by normal bovine anterior pituitary-derived cells. Initial purification of the acid-soluble TGF from concentrated conditioned medium was achieved by Bio-Gel P-60 gel filtration (apparent molecular mass of 9 kDa). After the Bio-Gel step, three different steps of reverse-phase fast-protein liquid chromatography on the same Pharmacia C18 column, using linear acetonitrile gradients, gave complete purification. The ion-pairing agents used in the three consecutive steps were: 0.1% trifluoroacetic acid, 0.13% heptafluorobutyric acid, and again, 0.1% trifluoroacetic acid at a shallower gradient. Homogeneity was confirmed by reverse-phase high performance liquid chromatography, and by polyacrylamide gel electrophoresis, where TGF visualization was facilitated by autoradiography of 125I-TGF. The 125I-TGF bound to epidermal growth factor (EGF) receptors and after elution ran identically to the starting material. The molecular mass of TGF is 6 kDa by polyacrylamide gel electrophoresis and 6.6 kDa by amino acid analysis. The amino acid composition of bovine TGF is similar to that of rat or human alpha TGF and distinct from epidermal growth factor. Colony-stimulating activity was lost after purification, but the TGF retained its ability to stimulate thymidine uptake by quiescent cells. This mitogenic activity could be blocked completely by anti-EGF-receptor monoclonal antibodies, indicating that the activity was mediated through the EGF-receptor.