Conservation of molecular structure of DNA polymerase beta in vertebrates probed by tryptic peptide mapping

DNA polymerase beta's from mouse myeloma, chick embryo, and cherry salmon testis were all composed of a single polypeptide of about 40K daltons as judged by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified enzyme preparations. Although the enzyme from bullfrog ovary was not fully purified, its molecular weight was estimated to be the same as that of the chick enzyme by immunological detection after electrophoresis. All the enzymes tested cross-reacted immunologically with the antibody against chick DNA polymerase beta, indicating that they have a common molecular structure, at least in part. Two-dimensional maps of radioiodinated tryptic peptides directly showed the presence of highly conserved amino acid sequences among mouse, chick, and cherry salmon enzymes. This conserved structure is thought to be essential for the enzyme activity, which is very similar among all these vertebrates.     

Purification, subunit structure, and DNA binding properties of the mouse oxysterol receptor

A cytosolic receptor protein for oxygenated sterols, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol biosynthesis, has been purified from mouse L cell cytosol greater than 3,600-fold in its undenatured form and to apparent homogeneity upon further electrophoresis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified 7.5 S receptor appears to be a dimer with similar or identical subunits of Mr 95,000. Proteolytic cleavage by an endogenous factor(s) gives rise to a 4.2 S form of the receptor which is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a heterogeneous mixture of ligand binding fragments of Mr 30,000-60,000. This 4.2 S form of the receptor retains high affinity for the oxysterol ligand and exhibits a more rapid oxysterol binding rate than the 7.5 S form. The 7.5 S form of the receptor binds to DNA-cellulose at low salt concentrations at neutral pH, and its affinity increases at low pH or in the presence of Zn2+. Receptor preparations from mouse liver were purified approximately 900-fold by the same purification procedure, but this was accompanied by conversion of the 7.5 S liver receptor to a approximately 4 S form, Mr approximately 55,000.     

Isolation and characterization of ciliary neurotrophic factor from rabbit sciatic nerves

Ciliary neurotrophic factor (CNTF) has been purified 35,000-fold to homogeneity from rabbit sciatic nerves using its ability to promote the survival of chick embryo ciliary ganglion neurons as the bioassay. The purification involved a combination of acid treatment, ammonium sulfate fractionation, hydrophobic interaction chromatography, chromatofocusing, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reversed-phase high performance liquid chromatography. Overlapping peptide sequences were obtained which accounted for 49% of the primary structure of the molecule. This information was used to prepare synthetic peptides in order to elicit antibodies. Purified CNTF exhibited two major and several minor bands between 24 and 22 kDa on silver-stained sodium dodecyl sulfate-polyacrylamide electrophoresis gels. All of the molecular forms were immunostained in Western blots by antiserum to synthetic peptides. The peptide sequences also provided a basis for cloning and expression of the rabbit CNTF gene (Lin, L-F. H., Mismer, D., Lile, J. D., Armes, L. G., Butler, E. T., III, Vannic, J. L., and Collins, F. (1989) Science 246, 1023-1025) confirming that the protein purified as reported here is CNTF.     

Biophysical characterization of the purified alpha 1-adrenergic receptor and identification of the hormone binding subunit

The alpha 1-adrenergic receptor has been solubilized in active form from rat hepatic membranes with the nonionic detergent, digitonin, and purified by affinity and gel filtration chromatography to homogeneity with a specific activity of 14,400 pmol/mg of protein. The affinity chromatographic steps of the purification procedure were achieved by the use of a newly synthesized analog (2-[4(2-succinoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, CP-57,609) of the highly selective alpha 1-adrenergic antagonist, prazosin, immobilized via an amide linkage to agarose. The resulting purified receptor bound [3H]prazosin and a variety of adrenergic agents with the specificity, stereoselectivity, and affinities equivalent to those observed with membrane-bound and solubilized receptor preparations. The purified receptor.digitonin complex had a Stokes radius of 49 A and a sedimentation coefficient (s20w) of 7.1, as determined by AcA-34 gel filtration chromatography and sucrose gradient density centrifugation, respectively. Based on these hydrodynamic parameters, the calculated molecular weight of the receptor.digitonin complex was estimated at 147,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis following the final purification step revealed a single band of protein at 59,000 daltons from which [3H]prazosin binding activity could be recovered after renaturation of the receptor protein. These findings indicate that the protein purified from rat hepatic membranes is the hormone binding component of the alpha 1-adrenergic receptor and that the receptor molecule most likely contains more than one Mr = 59,000 subunit.     

Purification of A1 adenosine receptor from rat brain membranes

The A1 adenosine receptor from rat brain membranes has been purified about 50,000-fold to apparent homogeneity by sequential use of affinity chromatography on immobilized xanthine amine congener-agarose, hydroxylapatite chromatography, and reaffinity chromatography. The overall yield starting from the membranes was approximately 4%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified preparation gave a broad single band of an apparent molecular weight of 34,000 either by silver staining or autoradiogram after radioiodination. The purified receptor bound approximately 24 nmol of 8-cyclopentyl-1,3-[3H]dipropylxanthine/mg of protein with a dissociation constant of 1.4 nM. This maximum specific binding value is consistent with the expected theoretical specific activity (29.4 nmol/mg) for a protein with a molecular mass of 34,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. Affinity-labeling experiments using [3H]p-phenylenediisothiocyanate-xanthine amine congener showed that the Mr = 34,000 protein band contained the ligand-binding sites. The purified receptor gave a typical A1 adenosine receptor pharmacological specificity similar to that of unpurified receptor preparations.     

Purification and properties of glyoxysomal lipase from castor bean

The alkaline lipase in the glyoxysomes from the endosperm of young castor bean seedlings, an integral membrane component, was solubilized in deoxycholate:KCl and purified to apparent homogeneity. The molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 62,000 daltons. The enzyme reaction was markedly stimulated by salts and inhibited by detergents. Triricinolein, the endogenous storage lipid, was hydrolyzed by the purified enzyme which is therefore a true lipase. Treatment of intact glyoxysomes with trypsin strongly diminished the lipase activity but did not affect matrix enzymes. An antibody preparation raised in a rabbit against the purified enzyme inhibited the purified enzyme and that in glyoxysomal membranes.     

Purification of the receptor for nerve growth factor from A875 melanoma cells by affinity chromatography

The receptor for nerve growth factor (NGF) has been purified to near homogeneity from octylglucoside extracts of A875 melanoma cell membranes by the use of repetitive affinity chromatography on NGF-Sepharose. Elution of purified receptor (NGF receptor) was accomplished with 0.15 M NaCl, pH 11.0, containing phosphatidylcholine and octylglucoside. Chromatography on two columns of NGF-Sepharose yielded a 1500-fold purification of the receptor, as assessed by 125I-NGF binding, and permitted recovery of 9% of the total binding activity in the soluble extract. Scatchard analysis of equilibrium binding of 125I-NGF provided similar Kd values for NGF receptors in soluble extracts of A875 membranes (2.2 nM) and with purified NGF receptor (3.1 nM). Examination of NGF receptor after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two major peptides, of Mr = 85,000 and Mr = 200,000. Affinity labeling experiments, done with 125I-NGF and A875 cells, soluble extracts of A875 cell membranes, and purified receptor, show that both of these components of the NGF receptor can be specifically cross-linked to 125I-NGF.     

Identification of a complex of the three forms of the rat liver asialoglycoprotein receptor

We have generated antibodies against synthetic peptides which represent the carboxyl terminus of either the major, or the two minor, forms of the rat hepatic lectin which recognizes galactose-terminated glycoproteins (asialoglycoproteins). The antibodies were shown to be specific for the form of the lectin containing the immunizing peptide sequence by the following: reaction with purified lectin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoprecipitation of sodium dodecyl sulfate-denatured lectin, immunoprecipitation of lectin synthesized in vitro. These antibodies, however, precipitated all three rat hepatic lectin forms from nonionic detergent extracts of hepatocytes labeled with 125I via the lactoperoxidase catalyzed technique. A similar result was obtained if antibody was bound to intact cells prior to solubilization with detergent and collection of the immune complexes. We conclude that at least the plasma membrane-associated fraction of the rat hepatic lectin forms exists as a heterotypic complex.     

Structural characterization of cardiac protein phosphatase with a monoclonal antibody. Evidence that the Mr = 38,000 phosphatase is the catalytic subunit of the native enzyme(s)

The native structures of protein phosphatases have not been clearly established. Several tissues contain high molecular weight enzymes which are converted to active species of Mr approximately 35,000 by denaturing treatments or partial proteolysis. We have used a monoclonal antibody directed against purified bovine cardiac Mr = 38,000 protein phosphatase to determine whether this species is the native catalytic subunit or a proteolytic product of a larger polypeptide. Monoclonal antibody was obtained from a cloned hybrid cell line produced by the fusion of Sp2 myeloma cells with spleen cells from a mouse immunized with phosphatase coupled to hemocyanin. This antibody was specific for the Mr = 38,000 phosphatase as determined by immunoblot analysis of purified enzyme or cardiac tissue extracts after native or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single immunoreactive protein of Mr = 38,000 was present in cardiac tissue extracts including extracts prepared from freeze-clamped rat heart rapidly denatured in hot sodium dodecyl sulfate buffer. Precipitation of cardiac extract with 80% ethanol did not alter the Mr of the phosphatase nor did it liberate new immunoreactive material not observed in the extract. Ethanol precipitation caused the dissociation of both phosphatase activity and immunoreactivity from a high Mr form to a form of Mr between 30,000 and 40,000. An immunoreactive protein of Mr = 38,000 was identified in several bovine and rat tissues as well as tissues from rabbits, mice and chickens and human HT-29 cells. From these data we conclude that the Mr = 38,000 cardiac phosphatase is a native catalytic subunit of higher molecular complexes which are dissociated by ethanol precipitation. A very similar, or identical, protein is present in several tissues and species suggesting that this catalytic subunit is a ubiquitous enzyme important in many dephosphorylation reactions.     

Purification of the insulin receptor from human placental membranes

Insulin receptors were purified from human placental microsomal membranes by solubilisation with Triton X-100 followed by Sepharose 6B chromatography, phosphate gradient elution from hydroxyapatite and affinity chromatography on concanavalin A-Sepharose. 2000-fold purification was achieved with 63% overall recovery. The purified receptor gave a single band on 3.75% polyacrylamide (0.1% Triton X-100) gel electrophoresis. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis there was a major band at 75,000 and a minor band at 80,000 daltons. The purified receptor rechromatographed on Sepharose 6B with an apparent molecular weight of 300,000.     

Purification and characterization of arginase from Neurospora crassa

We have purified an enzymatically active form of arginase from a wild-type strain of Neurospora crassa to homogeneity. The enzyme has a subunit molecular weight of 38,300 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein migrated as a hexamer during gel-filtration chromatography with an apparent molecular weight of 266,000. The enzyme exhibited hyperbolic kinetics at pH 9.5 with an apparent Km for arginine of 131 mM. Antiserum was prepared against the purified enzyme and used to demonstrate the existence of three cross-reactive proteins in crude extracts of wild-type N. crassa. One of these proteins corresponded to the purified protein, whereas the other two were of molecular weights 41,700 and 26,800, respectively. Using the same antiserum, we found that rat liver, but not rat kidney, contains immunoreactive material. We also detected two proteins in extracts of Saccharomyces cerevisiae that were weakly cross-reactive with the antiserum. These data provide evidence for the existence of multiple forms of arginase in fungi as well as in mammals.     

Purification of atrial natriuretic peptide receptor from bovine lung. Evidence for a disulfide-linked subunit structure

The receptor for atrial natriuretic peptide (ANP) was purified to apparent homogeneity from bovine lung by a combination of detergent extraction, ammonium sulfate precipitation, and affinity chromatography on ANP-Affi-Gel 10. The Mr of the purified receptor is about 140,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After reduction, the protein migrated as a single band with an Mr near 70,000. NH2-terminal sequence analysis of the purified material revealed only one sequence, indicating that the ANP receptor is composed of two probably identical subunits held together by disulfide bond(s), although it remains possible that one of the subunits is blocked at the NH2 terminus. Antibody was produced to the nonreduced Mr = 140,000 species and shown to interact with detergent-solubilized forms of the lung and kidney ANP receptor.     

Purification and characterization of an estrogen-inducible membrane glycoprotein. Evidence that it is a transferrin receptor

A number of N-linked membrane glycoproteins are induced during chick oviduct differentiation. We have purified a major estrogen-inducible glycoprotein (Mr = 91,000) to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of partial NH2-terminal sequence data with membrane glycoproteins having similar Mr showed a limited homology with human and murine transferrin receptors. We observed that oviduct membranes contain estrogen-inducible transferrin receptor activity (Kd = 2-8 x 10(-8) M). Analytical purification of the putative receptor on an ovotransferrin-Affi-Gel affinity column and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals a protein of Mr, 180,000, which contains two disulfide-linked subunits of Mr 91,000. The receptor reacts very strongly with antibodies prepared against the 91-kDa glycoprotein on Western blots. Western blot analysis confirms that the 91-kDa glycoprotein is induced by estrogen. The protein has 2% total carbohydrate with Man, GlcNAc, Gal, GalNAc, and NeuAc in a molar ratio of 6:4:2:1:1. The protein contains at least one O-linked moiety. Analysis of the O-linked moiety by glycosidase digestions and gel filtration indicates there are sialo tetra- and trisaccharides and a neutral disaccharide(s). Labeled N-linked glycopeptides were prepared by pronase digestion, beta-elimination, and 3H-acetylation. The N-linked oligosaccharides include high mannose and complex neutral nonbisected biantennary types in an approximate ratio of 3:1 as determined by serial lectin affinity chromatography.     

Cell wall proteins from sugar beet cells in suspension culture

Several proteins were extracted from the purified cell walls of suspension-cultured sugar beet cells with 0.5% EDTA (pH 6.8) after prior extraction of the walls with 0.5% deoxycholate and then with 2 molar NaCl. Two abundant proteins (P-I and P-II protein) were separately purified to homogeneity by procedures that included fractionation with ammonium sulfate, column chromatography on DEAE-cellulose and butyl Toyopearl, and preparative polyacrylamide electrophoresis. P-I exists as a dimer of identical subunits, and P-II is composed of four different subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that quite different polypeptides are present in the culture medium and in the NaCl and EDTA extracts of the wall.     

A Remeasurement of the Molecular Weights of T-Even Bacteriophage Substructural Proteins

T-even bacteriophage substructural proteins were studied by using discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that tail fibers are composed of two major proteins of 155,000 and 120,000 daltons molecular weight and four minor proteins of 51,000, 38,000, 27,000, and 23,000 daltons. Tail tubes were composed of one predominant protein of 18,500 daltons and one minor protein of 35,000 daltons molecular weight. Tubular polyheads obtained from a T4D amber mutant and by treatment of T4B-infected cells with L-canavanine were also examined, and no significant differences were noted in the molecular weight of the P23 protein.     

Purification and properties of prostaglandin E1/prostacyclin receptor of human blood platelets

Activation of platelet adenylate cyclase by prostaglandin E1 or prostacyclin is initiated through the interaction of the agonists with the same receptors on membrane. Prostaglandin E1/prostacyclin receptors of human platelets were solubilized in buffer, containing 0.05% Triton X-100 and protease inhibitors. The soluble membrane protein was chromatographed on a DEAE-cellulose column and assayed by a microfiber filter by equilibrium binding technique. The active fractions eluted at 0.7 M KCl were pooled, and the receptors were purified to homogeneity by Sephadex G-200 gel filtration with an overall recovery of 30%. The isolated receptor was 2,200-fold purified over the starting platelets. As evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the receptor showed a molecular mass of 190,000 daltons and is composed of two nonidentical subunits with molecular masses of 85,000 and 95,000 daltons. The interaction of prostaglandin E1 with the purified receptor was rapid, saturable, reversible, and highly specific. Among all prostaglandins tested, only prostacyclin was capable of displacing [3H]prostaglandin E1 bound to the receptor. Scatchard analysis of [3H]prostaglandin E1 binding to the purified receptor suggested the presence of a single class of high affinity binding sites (Kd = 9.8 nM) and a second population of low affinity binding sites (Kd = 0.7 microM) in the same protein molecule. Incubation of the purified receptor with platelets stripped of the receptor by washing with low concentrations of Triton X-100 efficiently restored the ability of prostaglandin E1 and prostacyclin to activate adenylate cyclase in these cells.     

Identification of a 55-kDa high-affinity calmodulin-binding protein from Electrophorus electricus

A high-affinity calcium-dependent calmodulin-binding protein (CaMBP) has been isolated from Electrophorus electricus main electric organ. This 55-kDa CaMBP has been purified to homogeneity by ion exchange and calmodulin-Sepharose affinity chromatography and electrophoretic elution from preparative sodium dodecyl sulfate-polyacrylamide gels. Antibodies against the 55-kDa CaMBP were raised in sheep and were affinity purified. A 47-kDa high-affinity CaMBP species was demonstrated by limited protease digestion and immunoblot analysis to be derived from the 55-kDa CaMBP. The 55-kDa CaMBP has also been isolated from skeletal muscle. It is not detectable by immunoblot analysis in nonexcitable tissues. Characterization of the 55-kDa high-affinity calmodulin-acceptor protein may further elucidate the role of calcium-calmodulin in the regulation of bioelectricity.     

Characterization of Saccharopolyspora erythraea cytochrome P-450 genes and enzymes, including 6-deoxyerythronolide B hydroxylase.

Previous studies of erythromycin biosynthesis have indicated that a cytochrome P-450 monooxygenase system is responsible for hydroxylation of 6-deoxyerythronolide B to erythronolide B as part of erythromycin biosynthesis in Saccharopolyspora erythraea (A. Shafiee and C. R. Hutchinson, Biochemistry 26:6204-6210 1987). The enzyme was previously purified to apparent homogeneity and found to have a catalytic turnover number of approximately 10(-3) min-1. More recently, disruption of a P-450-encoding sequence (eryF) in the region of ermE, the erythromycin resistance gene of S. erythraea, produced a 6-deoxyerythronolide B hydroxylation-deficient mutant (J. M. Weber, J. O. Leung, S. J. Swanson, K. B. Idler, and J. B. McAlpine, Science 252:114-116, 1991). In this study we purified the catalytically active cytochrome P-450 fraction from S. erythraea and found by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis that it consists of a major and a minor P-450 species. The gene encoding the major species (orf405) was cloned from genomic DNA and found to be distinct from eryF. Both the orf405 and eryF genes were expressed in Escherichia coli, and the properties of the proteins were compared. Heterologously expressed EryF and Orf405 both reacted with antisera prepared against the 6-deoxyerythronolide B hydroxylase described by Shafiee and Hutchinson (1987), and the EryF polypeptide comigrated with the minor P-450 species from S. erythraea on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. In comparisons of enzymatic activity, EryF hydroxylated a substrate with a turnover number of 53 min-1, whereas Orf405 showed no detectable activity with a 6-deoxyerythronolide B analog. Both enzymes showed weak activity in the O-dealkylation of 7-ethoxycoumarin. We conclude that the previously isolated 6-deoxyerythronolide B hydroxylase was a mixture of two P-450 enzymes and that only the minor form shows 6-deoxyerythronolide B hydroxylase activity.     

Myelin Basic Protein Is an Endogenous Inhibitor of the High-Affinity Cannabinoid Binding Site in Brain

Radioligand binding studies with the water-soluble cannabinoid [3H]5'-trimethylammonium delta 8-tetrahydrocannabinol ([3H]TMA) have revealed a saturable high-affinity site in brain that is specific for cannabinoids. To determine whether endogenous compounds of brain might act upon the site physiologically, we sought inhibitors in extracts of brain. An endogenous inhibitor has been purified to homogeneity by acid extraction of rat brain followed by adsorption to a reverse-phase matrix and gel filtration chromatography. The purified inhibitor has a subunit molecular mass of 14,500 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of [3H]TMA binding by the purified inhibitor occurs with a Ki of about 4 nM in a noncompetitive manner. The molecular weight, abundance, and extraction properties are the same as a species of myelin basic protein (MBP). The MBPs of rat, rabbit, pig, and cow also inhibit [3H]TMA binding noncompetitively with similar potencies. The purified inhibitor comigrates with rat MBP-small form on SDS-PAGE, has a similar amino acid composition, and is recognized by antibody directed against MBP. Studies of fragments of rabbit MBP suggest that the determinants of affinity for the [3H]TMA site are contained primarily within the C-terminal half of the rabbit MBP. Synthetic polycationic peptides such as polylysine and polyarginine mimic the effects of MBP, suggesting that the high-affinity cannabinoid binding site recognizes large polycations. The identification of the endogenous inhibitor of [3H]TMA binding as MBP suggests that MBP interacts physiologically with the high-affinity cannabinoid site.