Purification and properties of saccharopine dehydrogenase (glutamate forming) in the Saccharomyces cerevisiae lysine biosynthetic pathway.

Saccharopine dehydrogenase (glutamate forming) of the biosynthetic pathway of lysine in Saccharomyces cerevisiae was purified 1,122-fold by using acid precipitation, ammonium sulfate precipitation, DEAE-Sepharose, gel filtration, and Reactive Red-120 agarose chromatography. The enzyme exhibited a native molecular size of 69,000 daltons by gel filtration and consisted of a single 50,000-dalton polypeptide based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was readily denatured by exposures to temperatures exceeding 46 degrees C. The pH optimum for the reverse reaction was 9.5. The apparent Kms for L-saccharopine and NAD+ were 2.32 and 0.054 mM, respectively. The enzyme was inhibited by mercuric chloride but not by carbonyl or metal complexing agents.     

Purification and partial amino acid sequence of a bovine cartilage-derived collagenase inhibitor

An inhibitor of mammalian collagenase from bovine scapular cartilage has been purified to homogeneity. The inhibitor, extracted from cartilage using 2 M NaCl, was applied to an A-1.5m gel filtration column. Inhibitor eluted at an apparent Mr of 28,000. Further purification was achieved by ion exchange chromatography, gel filtration, and reverse-phase high performance liquid chromatography. A purification of greater than 1,000-fold was achieved. The inhibitor was judged homogeneous by the appearance of a single band on a silver-stained 15% sodium dodecyl sulfate-polyacrylamide gel. Reduced inhibitor had an Mr of 27,400, unreduced inhibitor had an Mr of 23,900. NH2-terminal sequence data were obtained for the first 45 residues. The bovine cartilage-derived inhibitor exhibits greater than 65% homology over the first 23 residues with a collagenase inhibitor purified from human skin fibroblasts maintained in cell culture. This is the first demonstration that collagenase inhibitors extracted directly from tissue may be similar to those obtained from culture medium.     

Subunit structure and properties of the glycogen-bound phosphoprotein phosphatase from skeletal muscle

A high molecular weight phosphoprotein phosphatase was purified approximately 11,000-fold from the glycogen-protein complex of rabbit skeletal muscle. Polyacrylamide gel electrophoresis of the preparation in the absence of sodium dodecyl sulfate showed a major protein band which contained the activity of the enzyme. Gel electrophoresis in the presence of sodium dodecyl sulfate also showed a major protein band migrating at 38,000 daltons. The sedimentation coefficient, Stokes radius, and frictional ratio of the enzyme were determined to be 4.4 S, 4.4 nm, and 1.53, respectively. Based on these values the molecular weight of the enzyme was calculated to be 83,000. The high molecular weight phosphatase was dissociated upon chromatography on a reactive red-120 agarose column. The sedimentation coefficient, Stokes radius, and frictional ratio of the dissociated enzyme (termed monomer) were determined to be 4.1 S, 2.4 nm, and 1.05, respectively. The molecular weight of the monomer enzyme was determined to be 38,000 by polyacrylamide gel electrophoresis. Incubation of the high molecular weight phosphatase with a cleavable cross-linking reagent, 3,3'-dithiobis(sulfosuccinimidyl propionate), showed the formation of a cross-linked complex. The molecular weight of the cross-linked complex was determined to be 85,000 and a second dimension gel electrophoresis of the cleaved cross-linked complex showed that the latter contained only 38,000-dalton bands. Limited trypsinization of the enzyme released a approximately 4,000-dalton peptide from the monomers and dissociated the high molecular weight phosphatase into 34,000-dalton monomers. It is proposed that the catalytic activity of the native glycogen-bound phosphatase resides in a dimer of 38,000-dalton subunits.     

Human epidermal transglutaminase. Preparation and properties.

A transglutaminase from human hair follicle-free epidermis was purified to homogeneity using gel filtration and ion exchange chromatography. The enzyme had an apparent Mr = 51,000 +/- 2,000 by sodium dodecyl sulfate electrophoresis, 100,000 +/- 5,000 by discontinuous gel electrophoresis, and 50,000 +/- 2,000 by gel filtration in Bio-Gel A-0.5m agarose. The enzyme cross-linked Factor XIII-free fibrinogen forming gamma dimers and alpha polymers. Either calcium or strontium was necessary for enzyme activity. In the presence of calcium, enzyme activity was increased by heating at 56 degrees or by treating with dimethylsulfoxide. Activation required calcium and occurred in the presence of serine protease inhibitors. The activated and native enzyme had apparently identical mobilities in acrylamide disc electrophoresis and sodium dodecyl sulfate electrophoresis. The Km values for two substrates in the reaction, casein and putrescine, were very similar for the native and the activated enzyme. The activated enzyme had a larger elution volume on Bio-Gel A-0.5m in the presence of calcium than did the native enzyme. The detailed mechanism of activation remains to be determined.     

Purification of follitropin receptor from bovine calf testes

Follitropin (FSH) receptors were solubilized from pure light membranes of bovine calf testis, using an optimum detergent to protein ratio of 0.01. The soluble FSH receptor fraction was gel filtered through Sepharose 6B to isolate an active fraction (6B-Fr-1) which behaved as a complex of FSH receptor and Gs protein. The 6B-Fr-1 was concentrated by ultrafiltration and further purified by sequential Sepharose 4B gel filtration, DEAE-cellulose chromatography (to separate the receptor from Gs protein), and wheat germ lectin affinity chromatography. The purified receptor had an FSH-binding capacity of approximately 3.47 nmol/mg of protein with a Kd of 1.9 X 10(-10) M. Yield was 526 micrograms/11.5 kg tested. Radioiodinated, as well as unlabeled purified FSH receptor, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single major band of Mr approximately 240,000. This band was not affected by 8 M urea treatment prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but treatment with dithiothreitol induced the loss of the 240-kDa band, with appearance of an Mr approximately 60,000 band. The availability of highly purified, stable FSH receptor should allow direct studies on its structure-function relationships.     

Purification and characterization of the sesquiterpene cyclase trichodiene synthetase from Fusarium sporotrichioides

The sesquiterpene cyclase, trichodiene synthetase, has been purified from a supernatant fraction of Fusarium sporotrichioides by hydrophobic interaction, anion exchange, and gel filtration chromatography. Purified enzyme had a specific activity 15-fold higher than that previously reported for preparations of terpene cyclases. Molecular weight determinations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography indicated the enzyme to be a dimer with a subunit of Mr 45,000. The requirement of Mg2+ (Km 0.1 mM) for activity could be partially substituted with Mn2+ at a concentration of 0.01 mM, but higher concentrations of Mn2+ were inhibitory. Maximum activity was observed between pH 6.75 and pH 7.75. The Km for farnesyl pyrophosphate was 0.065 microM.     

The purification and characterization of a phospholipase A in hamster heart cytosol for the hydrolysis of phosphatidylcholine

Phospholipases A1 and A2 catalyze the hydrolysis of acyl groups of phospholipids at C-1 and C-2, respectively. These phospholipases are important in phospholipid catabolism and the remodeling of the acyl groups of phospholipids. Phospholipase A from hamster heart cytosol was purified by a combination of ion-exchange and gel filtration chromatography. The purity of the enzyme was assessed by nondenaturing polyacrylamide gel electrophoresis, two-dimension polyacrylamide gel electrophoresis, and immunological studies. The purified enzyme exhibited both phospholipase A1 and A2 activities toward phosphatidylcholine and had the ability to hydrolyze the acyl groups of phosphatidylethanolamine. However, the enzyme was not active toward lysophosphatidylcholine, diacylglycerol, or triacylglycerol. By Sepharose 6B chromatography, the molecular weight of the purified enzyme was estimated to be 140,000. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the enzyme was composed of identical Mr 14,000 subunits. At least six subunits in the native enzyme could be cross-linked by dimethyl suberimidate. Both phospholipase A1 and A2 activities showed similar pH profiles, exhibited no absolute requirements for divalent metallic cations, but displayed a high degree of specificity for the acyl groups of phosphatidylcholine at both C-1 and C-2. The Km of phospholipases A1 and A2 for 1-palmitoyl-2-arachidon-ylglycerophosphocholine was found to be identical (0.5 mM).     

Purification of the secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum.

The secretor-type beta-galactoside alpha 1----2-fucosyltransferase from human serum was purified by hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, and affinity chromatography on GDP-hexanolamine-Sepharose. Final purification of the enzyme was achieved by high pressure liquid chromatography gel filtration and resulted in a homogeneous protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled protein. The native enzyme appears as a molecule of apparent Mr 150,000 as determined by gel filtration high pressure liquid chromatography. The apparent Mr of the enzyme resolved in the presence of beta-mercaptoethanol by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be 50,000, indicating a multisubunit structure of the enzyme. Secretor-type alpha 1----2-fucosyltransferase is a glycoprotein as determined by WGA binding properties. A comparison of the Mr of the native blood group H gene encoded with the secretor-type beta-galactoside alpha 1----2-fucosyltransferases as well as comparison of subunit Mr for both enzymes suggests structural similarity. The alpha 1----2 linkage formed between alpha-L-fucose and terminal beta-D-galactose by the purified H- and secretor-type alpha 1----2-fucosyltransferases was determined by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide products. The substrate specificity and Km values calculated from the initial rate using various oligosaccharide acceptors showed that purified enzymes differ primarily in affinity for phenyl-beta-D-galactopyranoside and GDP-fucose as well as type 1 (Gal beta 1----3GlcNAc), 2 (Gal beta 1----4GlcNAc), and 3 (Gal beta 1----3GalNAc) oligosaccharide acceptors. The secretor-type alpha 1----2-fucosyltransferase shows significantly lower affinity than the H enzyme for phenyl-beta-D-galactopyranoside and GDP-fucose as well as for type 2 oligosaccharide acceptors. On the contrary, type 1 and 3 oligosaccharide acceptors are preferentially utilized by the secretor-type enzyme as compared with the H enzyme. The enzymes also differ in several physicochemical properties, implying nonidentity of the two enzymes (Sarnesto, A., K?hlin, T., Thurin, J., and Blaszczyk-Thurin, M. (1990) J. Biol. Chem. 265, 15067-15075).     

Purification and properties of a second enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62.

An enzyme (splitting enzyme 2) which catalyzes the splitting of carbon-mercury linkage of arylmercury compounds was found in extracts of mercury-resistant Pseudomonas K-62. This enzyme was purified about 725-fold by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-75 and diethylaminoethyl-cellulose. A purified preparation of the enzyme showed a single band in electrophoresis either on polyacrylamide or sodium dodecyl sulfate-containing polyacrylamide gels. The molecular weight of the enzyme was estimated to be 20,000 (determined by Sephadex G-75 gel filtration) 17,000 (determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis). The enzyme showed a Km of 180 micron and a Vmax of 3.1 mumol/min per mg for p-chloromercuribenzoic acid and a Km of 250 micron and a Vmax of 20 mumol/min per mg for phenylmercuric acetate. The optimum temperature and pH for the reaction were 40 degrees C and 5.0, respectively.     

Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography

Proteins from sarcoplasmic reticulum vesicles solubilized by a nonionic detergent were fractionated by use of a reactive red-120 agarose column. The Ca-ATPase was obtained in pure form by eluting the column with 400 microM adenyl 5'-yl imidodiphosphate, yielding an enzyme of almost twice the starting specific activity in a fraction containing half the initial protein. The conclusion that the ATPase comprises 50% of the sarcoplasmic reticulum vesicle protein agrees with estimates gained from densitometry using 7 1/2% Laemmli slab gels but not from densitometry using 7% Weber and Osborn slab gels. The mechanism of purification was found to be affinity chromatography, with the ATPase binding the reactive red-120 ligand in its nucleotide-binding site. The steady-state concentration of phosphorylated intermediate relative to the specific activity was found to be lower in the purified enzyme as compared to the starting vesicular enzyme.     

Purification and partial characterization of the Mr 30,000 integral membrane protein associated with the erythrocyte Rh(D) antigen

Erythrocytes bearing the Rh(D) antigen have an Mr 30,000 integral membrane protein which can be surface-labeled with 125I and can be quantitatively immunoprecipitated from Triton X-100-solubilized spectrin-depleted membrane vesicles. The 125I-labeled Rh(D)-associated protein was purified to radiochemical homogeneity from membrane skeletons solubilized in sodium dodecyl sulfate and urea by hydroxylapatite chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. The Rh(D)-associated protein was purified nearly 200-fold from 2 units of erythrocytes from DD individuals by employing similar methods on a large scale using the purified 125I-labeled Rh(D)-associated protein as a tracer. The product appeared to be greater than 95% pure and migrated as a diffuse band of Mr approximately 30,000-32,000 on silver-stained sodium dodecyl sulfate electrophoresis gels poured from 12% acrylamide. It is estimated that the Rh(D)-associated protein makes up approximately 0.5% of the original membrane protein. When concentrated, partially purified Rh(D)-associated protein forms dimers and larger oligomers which are stable in sodium dodecyl sulfate and urea. The Rh(D)-associated protein was protected from degradation when intact erythrocytes or inside out membrane vesicles were enzymatically digested. These studies indicate that the Mr 30,000 protein associated with the Rh(D) antigen is linked to the membrane skeleton, resides within the lipid bilayer with minimal extra- or intracellular protrusions, exists normally as an oligomer, and can be purified in denatured form.     

Cross-linking of iodine-125-labeled, calcium-dependent regulatory protein to the Ca2+-sensitive phosphodiesterase purified from bovine heart.

The calcium-dependent regulatory protein (CDR).Ca2+ sensitive cyclic nucleotide phosphodiesterase was purified to apparent homogeneity from bovine heart by using ammonium sulfate fractionation, DEAE-ceelulose chromatography, and CDR-Sepharose affinity chromatography. The enzyme was purifed 13 750-fold with a 10% yield and a specific activity of 275 mumol of cAMP min-1 mg-1. The purified enzyme ran as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 57 000. Phosphodiesterase activity was stimulated 10-fold by Ca2+ and CDR with half-maximal activation occurring at 9 ng/assay. [125I]CDR was cross-linked to the purified phosphodiesterase by using dimethyl suberimidate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross-linked products revealed a number of discrete 125I-labeled bands. The molecular weights of the cross-linked products indicate that the stoichiometry of the phosphodiesterase complex is A2C2, where A is the phosphodiesterase catalytic subunit and C is the calcium-dependent regulatory protein.     

Purification and characterization of phosphatidylinositol kinase from porcine liver microsomes

Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis.     

Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal muscle microsomal membranes

Integral membrane-associated arginine-specific mono-ADP-ribosyltransferase was purified from rabbit skeletal muscle microsomes. The ADP-ribosyltransferase was solubilized from the 100,000 x g pellet with 0.3% sodium deoxycholate and purified to greater than or equal to 95% homogeneity by successive DE52, concanavalin A-agarose, 3-aminobenzamide-agarose, and size-exclusion high-performance liquid chromatography (HPLC) steps in the presence of detergents. Two molecular weight forms of the enzyme were isolated and partially characterized. The apparent Mr of the alpha-form of the enzyme purified to greater than or equal to 95% homogeneity was approximately 39,000 +/- 500 as estimated by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr of the beta-form purified to greater than or equal to 80% homogeneity was 38,500 +/- 500. The rapid procedure resulted in a 200-fold purification for the alpha-form and a 645-fold purification for the beta-form, relative to the microsomal fraction. Positive identification of the enzyme was confirmed by utilizing a zymographic in situ gel assay and by HPLC assay of polyacrylamide gel slice incubations with an NAD and guanylhydrazone substrate. The specificity of the mono-ADP-ribosyltransferase zymographic assay was characterized by time course incubations, hydroxylamine sensitivity, 3-aminobenzamide inhibition, and histone dependence. The ADP-ribosyltransferase is inactivated by reducing agents.     

Stereoisomerism in plant disease resistance: induction and isolation of the 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone oxidoreductase, an enzyme introducing chirality during synthesis of isoflavonoid phytoalexins in pea (Pisum sativum L)

Treatment of pea seedlings with CuCl2 induced the activity of the enzyme NADPH:7,2'-dihydroxy-4',5'-methylenedioxyisoflavone oxidoreductase (DMIRase) that introduces (+) stereoisomerism in pisatin. DMIRase was purified approximately 7000 fold from CuCl2-treated pea seedlings to apparent homogeneity by a six-step process. The purification sequence included (NH4)2SO4 fractionation, gel filtration on AcA 44, chromatography on DEAE-Bio-Gel,phenyl-Sepharose CL-4B, and Reactive Red 120-agarose, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration and denaturing electrophoresis showed that the enzyme consisted of a single polypeptide chain with an Mr of 37,500. The pH optimum of DMIRase was determined to be 7.8. The enzyme showed apparent Michaelis constants of 20 microM for 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone and 58 microM for NADPH. The reaction product of the enzyme, sophorol, gave a distinct negative Cotton effect in the region 300-360 nm, which indicated 3S configuration of the molecule. Antibodies against the enzyme were raised in rabbits and characterized for specificity.     

Purification by affinity chromatography and physicochemical properties of the guanine-specific ribonuclease of Fusarium moniliforme

Ribonuclease F1, the guanine-specific ribonuclease of Fusarium moniliforme, was purified to homogeneity by a combination of ethanol fractionation, affinity chromatography and DEAE-cellulose column chromatography. The adsorbent for the affinity chromatography was synthesized by the coupling of periodate-oxidized guanosine 5'-monophosphate to aminohexyl agarose followed by sodium borohydride reduction. Ribonuclease F2, the minor component, was also purified to near homogeneity by the same procedure. Ribonucleases F1 and F2 had the same molecular weight (about 11,000) as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. They also showed the same amino acid composition and differed only in the isoelectric point: 4.10 for F1 and 3.96 for F2.     

Purification of the beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase from human serum.

A beta-N-Acetylglucosaminide alpha 1----3-fucosyltransferase was purified from human serum by ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-hexanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration. Gel filtration chromatography of the native enzyme revealed a Mr of 45,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein also appeared as a single molecular species of Mr 45,000. In contrast to the multisubunit beta-galactoside alpha 1----2-fucosyltransferases with an apparent Mr of 150,000, present in human serum, the native beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase is a monomer with a Mr of 45,000. The enzyme is glycosylated, as revealed by wheat germ agglutinin binding properties. The alpha 1----3 linkage formed by the enzyme between alpha-L-fucose and the penultimate beta-N-acetylglucosamine by the purified enzyme was confirmed by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide product. The specificity of the purified enzyme is restricted to type 2 structures, as revealed by its reactivity with different substrates and from the Km values calculated from the initial rate data using various oligosaccharide acceptors. The enzyme has the ability to utilize the N-acetyl-beta-lactosamine determinant (Gal beta 1----4GlcNAc) and the sialylated (NeuAc alpha 2----3Gal beta 1----4GlcNAc) and fucosylated (Fuc alpha 1----2Gal beta 1----4GlcNAc) derivatives of N-acetyl-beta-lactosamine and thus is distinct from both the human Lewis gene-encoded enzyme and the alpha 1----3-fucosyltransferase of the myeloid cell type.     

Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor

A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M.     

Second-step transfer of bacteriophage T5 DNA: purification and characterization of the T5 gene A2 protein.

Second-step transfer of bacteriophage T5 DNA requires the function of the T5 pre-early proteins A1 and A2. We have isolated and characterized the gene A2 protein as part of an effort to determine the mechanism of second-step transfer. The A2 protein was purified by DNA-cellulose column chromatography followed by gel filtration and ion-exchange column chromatography. The A2 protein's identity was confirmed by two-dimensional gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer gel filtration in 6 M guanidine hydrochloride demonstrated a molecular weight of 15,000 for the A2 polypeptide. Migration of the A2 protein through gel filtration columns under nondenaturing conditions, in combination with sedimentation behavior, indicated dimerization of the A2 polypeptide. The existence of the A2 dimer was confirmed by protein cross-linking with dimethyl suberimidate and analysis of the cross-linked proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition, degree of polymerization, DNA-binding ability, and physical characteristics of the T5 gene A2 protein are consistent with a function of the A2 protein in DNA transfer.     

Transforming growth factors from a human tumor cell: characterization of transforming growth factor beta and identification of high molecular weight transforming growth factor alpha

Intracellular transforming growth factors (TGFs) were extracted from a human rhabdomyosarcoma cell line and purified to apparent homogeneity by using gel filtration, cation-exchange, and high-performance liquid chromatography. Two types of transforming growth factor activities, TGF-alpha and TGF-beta, were detected. The intracellular polypeptides which belonged to the TGF-alpha family required TGF-beta for full activity in inducing nonneoplastic normal rat kidney fibroblasts to grow in soft agar. These peptides also bound to the membrane receptor for epidermal growth factor. As determined by sodium dodecyl sulfate-polyacrylamide gels, the apparent molecular weight of these intracellular TGF-alpha's was 18 000. Intracellular TGF-beta required either epidermal growth factor or TGF-alpha for stimulation of soft agar growth. The intracellular TGF-beta was purified to homogeneity as judged by a single peak after reverse-phase high-performance liquid chromatography and a single band on a sodium dodecyl sulfate-polyacrylamide gel. The intracellular TGF-beta from the human tumor cell line was similar in all respects tested (migration on sodium dodecyl sulfate-polyacrylamide gels, stimulation of soft agar growth, binding to the membrane receptor for TGF-beta, and amino acid composition) to intracellular TGF-beta from normal human placenta.