Immunoprecipitation of the lutropin/choriogonadotropin receptor from biosynthetically labeled Leydig tumor cells. A 92-kDa Glycoprotein

The structure of the lutropin/choriogonadotropin (LH/CG) receptor has been studied by immunoprecipitating the receptor from biosynthetically labeled cultured Leydig tumor cells (designated MA-10). This was performed by binding human choriogonadotropin (hCG) to the labeled cells, solubilizing the hormone-receptor complex, partially purifying the complex by lectin chromatography, and immunoprecipitating the complex with an antibody that recognizes receptor-bound hCG. The conditions used for the release of the radiolabeled receptor from the immunoprecipitate and the subsequent analysis of this material on sodium dodecyl sulfate gels allowed us to determine directly the structure of the free (not hormone-occupied) LH/CG receptor. From experiments using cells labeled with [35S]methionine and [35S]cysteine, we show that the LH/CG receptor is composed of a single polypeptide chain that migrates as a 92-kDa protein on sodium dodecyl sulfate gels whether analyzed in the absence or presence of reducing agents. Other studies presented demonstrate that the LH/CG receptor is a glycoprotein.     

Transformation in pneumococcus: existence and properties of a complex involving donor deoxyribonucleate single strands in eclipse.

Donor deoxyribonucleic acid (DNA) single strands exist in a complex during the eclipse phase in pneumococcal transformation. This eclipse complex exhibited specific physical properties distinct from those of both pure DNA single strands and native DNA. These included a lower affinity for diethylaminoethyl-cellulose and hydroxylapatite than that of single-strand DNA, faster sedimentation than the DNA chains that it contains, and a buoyant density in Cs2SO4 lower than that of native DNA. The complex was dissociated by treatments with sodium dodecyl sulfate, NaOH, guanidine-hydrochloride, chloroform, and proteinase K but was insensitive to ribonuclease.     

Purification of an active opioid-binding protein from bovine striatum

We report the purification to apparent homogeneity of an active opioid-binding protein solubilized from bovine striatal membranes. The purification was accomplished in two steps: affinity chromatography on beta-naltrexylethylenediamine (NED)-CH-Sepharose 4B followed by lectin affinity chromatography on wheat germ agglutinin-agarose. The ligand affinity-purified fraction exhibits stereospecific and saturable binding of opiates and is heat-sensitive. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the NED-purified material gave 6-8 bands by silver staining or autoradiography of radioiodinated material. Under nondenaturing conditions, the NED-purified material elutes in a molecular mass range between 300 and 350 kDa from gel exclusion chromatography on Ultrogel AcA-34. The specific activity of the affinity-purified fraction (800-1500 pmol/mg protein) is enriched 4000 to 7000-fold over that of the membrane-bound or unpurified soluble receptor. Further purification (10-20-fold) is achieved by chromatography of the NED eluate on wheat germ agglutinin-agarose. The eluted fraction shows a single protein (65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material is an acidic glycoprotein with a pI of 6.0-6.3 and binds opiates with a specific activity (12,000-15,000 pmol/mg) that is 65,000 to 75,000-fold greater (theoretical, 77,000-fold) than that of the membrane-bound or crude soluble receptors.     

Oxidation of fatty alcohol in the cotyledons of jojoba seedlings.

An externally accessible polypeptide has been purified from hepatoma tissue culture cells. The purification involves four steps: deoxycholate extraction of whole cells, isoelectric focusing of deoxycholate-insoluble material in the presence of 8 m urea and Triton X-100, hydroxylapatite chromatography in the presence of sodium dodecyl sulfate, and preparative acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The final preparation is homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by isoelectric focusing in polyacrylamide. The polypeptide has an apparent molecular weight of 55,000 and is labeled following in situ lactoperoxidase-catalyzed iodination of the hepatoma tissue culture cells. The polypeptide can also be labeled by growing cells in the presence of labeled amino acids, but is not labeled by growth in labeled sugars. The purified protein does not react with the periodate-Schiff reagent. Hence, it does not appear to be a glycoprotein that contains mannose, fucose, glucosamine, or sialic acids.     

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.     

Human blood group glycosyltransferase. II. Purification of galactosyltransferase

A galactosyltransferase, which converts blood group O red bloodcells to B-cells, was purfied to homogeneity from plasma of blood group B subjects. The stepwise purification procedures include: (a) column chromatography with CM-Sephadex, followed by ammonium sulfate fractionation; (b) Sephadex G-200 gel filtration; (c) column chromatogr,phy with DEAE-Sephadex; and (d) column chromatography with hydroxylapatite. The procedures provided about a 400,000-fold increase of specific activity with a 40 to 50% yield. Further purification of the enzyme was performed by small scale preparative acrylamide gel electrophoresis at pH 4.3. The final enzyme preparation showed a single protein band which coincided with enzyme activity, in acrylamide gel electrophoresis, and revealed a single protein band in sodium dodecyl sulfate-gel electrophoresis. Judging from the molecular weight, which was estimated by Sephadex gel filtration, and subunit size estimated by sodium dodecyl sulfate-gel electrophoresis, the enzyme is presumably in a dimeric form. The enzyme required Mn2+ for its activity and had a pH optimum at 7.0 to 7.5.     

Purification and partial characterization of a bacteriocin from Serratia marcescens

Bacteriocin JF246 has been purified from mitomycin C-induced Serratia marcescens cells by salt extraction, ammonium sulfate fractionation, and chromatography on QAE-Sephadex and SP-Sephadex. The purified material is homogeneous on polyacrylamide gel electrophoresis in the presence of 2% sodium dodecyl sulfate or 6 m urea. In the absence of these agents, the bacteriocin associates into aggregates which can be dissociated with 0.4 m NaCl. The bacteriocin is probably composed of a single subunit with a molecular weight of 64,000 daltons. Analytical studies show the bacteriocin to be essentially protein in nature containing less than one residue of glucose or phosphorus per 64,000 daltons.     

A simple chromatographic procedure for the purification of the D dimer fragment from crosslinked fibrin

An improved procedure for the purification of fragment D dimer derived from crosslinked plasma fibrin is described which entails chromatofocusing chromatography using PBE 94 and polybuffer 74, and gel chromatography on Sephacryl S-300. The procedure provides a preparation of D dimer which behaves as a single macromolecular entity with molecular weight 190,000 in sedimentation equilibrium studies. Only a single protein band is observed in polyacrylamide gel electrophoresis conducted in the presence or absence of sodium dodecyl sulfate, while patterns characteristic of gamma'-gamma' chains are observed under denaturing conditions after reduction of the preparation with beta-mercaptoethanol. The D dimer contains no demonstrable E antigen by a range of electrophoretic and immunologic techniques. Advantages of this method for obtaining D dimer in high yield include the use of plasma as starting material, the use of a simple lysis regimen in the presence of Ca2+, and the use of simple chromatographic techniques performed under nondenaturing conditions.     

Purification of BBF, a DNA-binding protein recognizing a positive cis-acting element in the mouse alpha 1(III) collagen promoter.

A positive cis-acting element, the B element, located between -83 and -61 in the mouse alpha 1(III) collagen promoter, binds a factor present in nuclear extracts of NIH 3T3 fibroblasts and HeLa cells. We have purified this factor using ion exchange chromatography, sequence-specific DNA affinity chromatography, and sodium dodecyl sulfate-polyacrylamide gel fractionation. The DNA sequence used for the affinity chromatography was a single-base substitution in the B element that increased the stability of the B element-protein complex by 50%. Purification of the B element-binding factor (BBF) by DNA affinity chromatography resulted in the apparent loss of most or all of the DNA-binding activity of this factor. The DNA-binding activity could, however, be reconstituted by combining two chromatographic fractions: the high-salt eluate and the column flow-through. When the partially purified high-salt eluate was size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with subsequent renaturation of gel fractions from guanidine HCl, the purified BBF (apparent molecular weight of about 95,000) bound to the B element with high affinity. These results suggest that during DNA affinity purification of BBF a factor that inhibits BBF DNA binding was co-eluted with BBF. This inhibition of BBF DNA binding was reversed by the addition of the DNA affinity column flow-through. The binding of BBF to the B element of the mouse alpha 1(III) collagen promoter is therefore an apparently complex process involving interactions between BBF and other protein factors.     

A method for efficient and selective recovery of membrane glycoproteins from concanavalin A-Sepharose using media containing sodium dodecyl sulfate and urea

Herpes-specific membrane glycoproteins were recovered from infected cells by incubating total homogenates with Con A-Sepharose in sealed plastic tubes. Following affinity binding of glycoproteins, subsequent washes with media containing high-salt concentrations followed by washes in 0.1% sodium dodecyl sulfate effectively removed nonglycoprotein contaminants. Glycoproteins were then eluted in high yield by heating the Con A-Sepharose-glycoprotein complex in medium containing 5% sodium dodecyl sulfate and 8 m urea. Eluates were placed directly onto sodium dodecyl sulfate-polyacrylamide gels for further analysis and purification of individual components. The procedure described here is convenient for simultaneously processing many different samples on either a large or small scale.     

Overproduction in Escherichia coli K-12 and purification of the TraJ protein encoded by the conjugative plasmid F

The TraJ protein encoded by the conjugative plasmid F has been designated an Escherichia coli K-12 envelope protein that participates in a mechanism of gene regulation. We have purified the TraJ protein, using an Flac::lambda traJ lysogen that overproduces the protein after heat induction of the prophage. Sufficient TraJ protein was synthesized within 40 min after induction to follow the purification by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Our purification exploited the observation that the TraJ protein remains insoluble after repeated Triton X-100/EDTA extractions of crude envelope fractions. The protein was then solubilized in sodium dodecyl sulfate at 60 degrees C and fractionated further by gel filtration and hydroxylapatite chromatography, both in the presence of sodium dodecyl sulfate. After hydroxylapatite chromatography, the protein was greater than 95% pure. The identity of the purified protein was confirmed by analysis of its NH2-terminal amino acid sequence, which was the same as that predicted from the partial nucleotide sequence of the traJ gene (Thompson, R., and Taylor, L. (1982) Mol. Gen. Genet. 188, 513-518). This analysis also indicated that the TraJ protein is localized in the cell without proteolytic modification of its NH2-terminus. We discuss the possible significance of these observations with respect to the cellular functions of the TraJ protein.     

Purification of the colicin I receptor

The colicin I outer membrane receptor was solubilized from the cell envelope of Escherichia coli K12 by extraction with Triton X-100 and purified to homogeneity by a combination of ion exchange and gel filtration chromatography as well as isoelectric focusing. The receptor was isolated as a single polypeptide and retained capacity to form a complex with pure colicin. The apparent molecular weight of the receptor as determined by polyacrylamide gel electrophoresis in sodium dodecy sulfate was 74,000 or 54,000 depending on whether the preparation was boiled or not in sodium dodecyl sulfate, respectively, prior to electrophoresis. Isoelectric focusing of the receptor in the presence of Triton X-100 revealed that the protein was slightly acidic (pI 4.75).     

Immunological identification of complex proteins resolved by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis.

Immunological identification of an antigen resolved from a protein complex by sodium dodecyl sulfate polyacrylamide gel electrophoresis has been attained. The identification is based on the formation of immunoprecipitin lines after the antigen diffuses laterally from acrylamide gel transverse slices into a surrounding agarose gel. This technique was designed for study of contractile and regulatory protein complexes of non-muscle cells where the scarcity of tissue precludes easy purification or high yield of muscle-like proteins. It complements double-gel immunodiffusion or immunoelectrophoresis and its use may be extended to other protein complexes.     

Isolation of a sodium dodecyl sulfate-insoluble transglutaminase substrate from liver plasma membranes

Rat liver plasma membranes contain transglutaminase activity and a large molecular weight protein complex which serves as a substrate for this enzyme. When plasma membranes were solubilized in sodium dodecyl sulfate and disulfide-reducing agents the transglutaminase substrate was recovered in the detergent-insoluble fraction. The insolubility of the complex suggested that it might be further studied by adsorbing membranes onto glass slides, then extracting with the detergent and reducing agent. After extraction, dark field light microscopy revealed numerous flattened sheets which varied in size from 4 to 12 micrometers. To confirm that these structures were the large molecular weight transglutaminase substrate, the plasma membranes were solubilized in sodium dodecyl sulfate and dithiothreitol and sedimented through a sucrose gradient containing the agent. The large molecular weight substrate was the only material found at the 1.11/1.23 g/cm3 interface. Microscopic examination showed the same structures previously observed on the glass slides. We conclude that the large molecular weight transglutaminase substrate is a sodium dodecyl sulfate-insoluble, morphologically distinct, protein complex. Due to its considerable size, nondissociable nature, and association with the lateral membrane, the sodium dodecyl sulfate-insoluble transglutaminase substrate may serve as a type of skeleton or scaffolding for this plasma membrane domain.     

Purification of a factor inhibiting differentiation from conditioned medium of nondifferentiating mouse myeloid leukemia cells

Mouse myeloid leukemic M1 cells are induced to differentiate by various differentiation inducers. Activity for inhibition of induction of differentiation of M1 cells (I-factor activity) was detected in conditioned medium of variant M1 cell clones that were resistant to differentiation inducers, and this I-factor activity was shown to be closely associated with resistance of the cells to differentiation inducers. In this work, the I-factor was purified to apparent homogeneity from conditioned medium of resistant M1 cells. The purification procedure consisted of ammonium sulfate precipitation, CM-Sepharose CL-6B, Sephadex G-200, reverse-phase high performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The factor was analyzed by radioiodination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography. The purified factor gave a single band of protein with a molecular weight of 68,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis which coincided with its biological activity. The concentration of I-factor required for 50% inhibition of dexamethasone-induced differentiation of M1 cells was 24 pM. At its effective concentration it had no effect on cell proliferation, and even at 1.2 nM it did not inhibit colony formation of normal bone marrow cells, suggesting that it was distinct from the inhibitor of normal precursors of macrophages and/or granulocytes.     

Sarcolectin: an interferon antagonist extracted from hamster sarcomas and normal muscles. Isolation, characterization, and purification

In the present work we show that sarcoma and normal hamster tissues contain a protein which agglutinates normal and transformed cells. The inhibition of agglutination by galacturonic acid and occasionally by fucose suggests a resemblance of this protein with vegetal lectins. When added 5 h after interferon, the crude semipurified and electrophoretically homogeneous preparations reduce within 20 h the antiviral state pre-established by interferon. These two biological tests have enabled us to monitor the subsequent purification steps. The isolation of the biologically active protein is greatly facilitated by its resistance to pepsin and nucleases, whereas its sensitivity to trypsin and Pronase suggests its proteinaceous character. Furthermore, the molecule is stable when heated 1-2 min at 100 degrees C in the presence or absence of sodium dodecyl sulfate. After pepsin treatment, Sephacryl G-200 gel filtration, and ion exchange chromatography on DEAE-cellulose, 25-40-fold purification can be obtained. When controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a double band (DEAE-cellulose sample) or single band (octyl-agarose sample) is detected in the 65,000-dalton region and no other contaminator is present. The eluted protein retains full biological activity when assayed by the degradation of the interferon-induced antiviral protection in the cell or titrated by cell agglutination.     

Isolation and characterization of methionine synthetase from human placenta

The cobalamin-dependent enzyme, methionine synthetase, has been purified approximately 1000-fold to apparent homogeneity from human placenta with a 19% recovery. The final two steps of the purification utilized two different affinity columns. The first was a N5-methyltetrahydrofolate-cystamine-agarose column, and the second was a S-adenosylhomocysteine-agarose column. The enzyme was eluted from the first affinity column by buffer containing reducing agent which released the folate and the enzyme while elution from the second affinity column was accomplished with buffer containing 0.5 M sodium chloride. Criteria for purity were the observations that single peaks of enzyme activity, protein, and cobalamin with an apparent molecular weight of 160,000 were obtained by gel filtration and that holomethionine synthetase contained 1 mol of cobalamin/mol of protein. Furthermore, analysis by high performance liquid chromatography using a molecular weight sizing column demonstrated a single peak of protein with a corresponding cobalamin peak. This single peak of protein was progressively converted to a second protein peak that was enzymatically inactive, and this conversion was associated with a directly proportional loss of enzyme activity and cobalamin from the first peak. Methionine synthetase appeared to have a molecular weight of 160,000 on unreduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and subunits of Mr 90,000, 45,000, and 35,000 on reduced sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis.     

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).     

Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting

DNA polymerase delta was isolated from human placenta and identified as such on the basis of its association with a 3'- to 5'-exonuclease activity. The association of the polymerase and exonuclease activities was maintained throughout purification and attempted separations by physical or electrophoretic methods. Moreover, ratios of the two activities remained constant during the purification steps, and both activities were inhibited by aphidicolin, oxidized glutathione, and N-ethylmaleimide. The purified enzyme had an estimated molecular weight of 172,000, on the basis of a Stokes radius of 53.6 A and a sedimentation coefficient of 7.8 S. On sodium dodecyl sulfate (SDS) gel electrophoresis, polymerase delta preparations contained a band of ca. 170 kilodaltons (kDa) as well as several smaller polypeptides. The 170-kDa polypeptide was identified as the largest polypeptide component in the preparation possessing DNA polymerase activity by an activity staining procedure following gel electrophoresis in the presence of SDS. Western blotting of DNA polymerase delta with polyclonal antisera also revealed a single 170-kDa immunoreactive polypeptide. Monoclonal antibodies to KB cell polymerase alpha inhibited placental polymerase alpha but did not inhibit DNA polymerase delta, while the murine polyclonal antisera to polymerase delta inhibited delta but not alpha. These findings establish the existence of DNA polymerase delta in a human tissue and support the view that both its polymerase and its exonuclease activities may be associated with a single protein.