Postnatal D2-CAM/N-CAM Sialylation State Is Controlled by a Developmentally Regulated Golgi Sialyltransferase

Abstract: Golgi-enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2-CAM/N-CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12-day-old animals the majority of the transferred [¹⁴C]sialic acid was found to be associated with the high-molecular-weight [>200 kilodaltons (kd)] form of D2-CAM/N-CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2-CAM/N-CAM polypeptides was also demonstrated in both 12-day and adult animals and transfer was evident only in the 180-kd and 115-kd components and not in the 140-kd component. In contrast, Golgi-enriched fractions prepared from adult animals showed little capability of heavily sialylating D2-CAM/N-CAM to any significant extent.     

Masking of epitopes in tissue sections

Summary Antisera to chicken brain antigen (CBA) isolated by hydroxyapatite chromatography from 8 M urea extracts following repeated extractions with phosphate buffer selectively decorate neurofilaments (NF) in neuronal perikarya, dendrites and axons. The antisera also reacted with GFA protein, the astrocyte-specific intermediate filament protein, as indicated by the adsorption of NF immunoreactivity following passage of the antisera through columns prepared with purified GFA protein. Moreover, the antisera stained the polypeptides of the NF triplet (70 kd, 150 kd, 200 kd) and GFA protein by the immunoblotting procedure. Monoclonal antibodies selectively decorating NF in tissue sections were isolated from a fusion of mouse myeloma cells with spleen cells of mice immunized with CBA. By the immunoblotting procedure the antibodies decorated the 150 kd NF polypeptide and GFA protein. No staining of glial filaments or any other structure on tissue sections was also observed with antibodies derived from another fusion strongly reacting with GFA protein on immunoblots. All antibodies (monoclonal and polyclonal) appeared to react with the same region of the GFA polypeptide as indicated by immunoblots of cleavage products.     

Synthesis and organization of vitellogenin and vitellin molecules from the land crab Potamon potamios

We have previously reported that vitellogenin (Vg) of some female animals contained four polypeptides with molecular mass of 181, 115, 105 and 85 kDa, whereas Vg of most animals contained three polypeptides with molecular mass of 115, 105 and 85 kDa. In the present investigation, we examined whether the 181 kDa polypeptide is the precursor of 115 and 105 kDa Vg and vitellin (Vn) polypeptides. Labeling studies, using [35S]methionine on normal vitellogenic animals, showed that the radioactivity was distributed first among the 181 and 85 kDa polypeptides. SDS-PAGE analysis of purified hemolymph Vg from eyestalk ablated female animals revealed in most animals two polypeptides with an apparent molecular mass of 181 and 85 kDa. These results from in vivo experiments corroborated the view that the 115 and 105 kDa Vg and Vn polypeptides are derived from heaviest 181 kDa polypeptide. In addition it was demonstrated that hepatopancreas and ovary of Potamon potamios incubated in vitro with [35S]methionine synthesized five polypeptides with apparent molecular mass of 224, 181, 115, 105, and 85 kDa while the hepatopancreas appeared to secrete the 181, 115, 105 and 85 kDa polypeptides. The major 115, 105 and 85 kDa polypeptides were found to be components of egg Vn, while the 224 kDa polypeptide was found to be minor component of Vg and Vn from hepatopancreas and ovary extracts, respectively. We conclude that the Vn polypeptides produced by ovary are similar to those produced by hepatopancreas.     

Purification and characterization of a protein tyrosine kinase from bovine spleen

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.     

Biochemical characterization of H9/25, an allospecificity encoded by the Ly-6 region

H9/25, an allospecificity encoded by the Ly-6 region, was biochemically characterized. It was sensitive to pepsin and heat treatment, but was resistant to periodate oxidation. Its apparent molecular weight was approximately 12 000 daltons by gel filtration. The antigenic molecule was partially purified by gel filtration and antibody affinity chromatography. The partially purified antigen molecule was radioiodinated, immunoprecipitated with monoclonal antibody H9/25, and analyzed by SDS-polyacrylamide gel electrophoresis. The autoradiograph showed the molecular weight of H9/25 to be approximately 15000 daltons under reducing conditions. These results indicate that H9/25 is a protein with a single polypeptide chain of 12000–15000 daltons molecular weight, and the antigenic specificity is carried by a peptide but not a carbohydrate moiety.     

Purification of the individual snRNPs U1, U2, U5 and U4/U6 from HeLa cells and characterization of their protein constituents.

A procedure is described for the purification of the individual major small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4/U6 from HeLa cells. The salient feature of the method is the combined usage of antibodies against 2,2,7-trimethylguanosine (m3G) and 6-methyladenosine (m6A) for differential immune affinity chromatography of the snRNPs. While anti-m3G affinity columns allow the separation of snRNPs U1, U2 and U5 from U4/U6 RNPs, anti-m6A antibodies selectively react with snRNPs U2 and U4/U6. Our technique further incorporates immune affinity chromatography of snRNPs with antibodies against snRNP proteins in addition to ion exchange chromatography. The procedure avoids the usage of denaturing agents, so as to maintain the native structure of the particles. This is mainly provided for by the possibility of eluting the anti-m3G and anti-m6A bound snRNPs with excess of the respective nucleosides. We have so far identified 12 polypeptides as constituents of the major snRNPs U1 to U6. Seven proteins of approximate mol. wts 29 kd (B'), 28 kd (B), 16 kd (D), 15.5 kd (D'), 12 kd (E), 11 kd (F) and 9 kd (G) were present in each of the individual snRNPs U1, U2, U5 and U4/U6. In addition to the common proteins, U1 RNPs contain three unique polypeptides of mol. wts 70 kd, 34 kd (A) and 22 kd (C). U2 RNPs are characterized by the presence of a 33-kd and a 28.5-kd protein, denoted A' and B". We could not detect any unique polypeptide confined to the purified snRNPs U5 or U4/U6.(ABSTRACT TRUNCATED AT 250 WORDS)     

3' cleavage and polyadenylation of mRNA precursors in vitro requires a poly(A) polymerase, a cleavage factor, and a snRNP

We have separated and purified three factors from HeLa cell nuclear extracts that together can accurately cleave and polyadenylate pre-mRNAs containing the adenovirus L3 polyadenylation site. One of the factors is a poly(A) polymerase with a molecular weight of approximately 50-60 kd. The second activity is a cleavage factor with a native molecular weight in the range of 70-120 kd. The third component is a factor (cleavage and polyadenylation factor, CPF) that is needed for the cleavage reaction and, in addition, confers specificity to the poly(A) polymerase activity; the native molecular weight of CPF is approximately 200 kd. Poly(A) polymerase together with CPF is sufficient to specifically polyadenylate pre-mRNA substrates that have been precleaved at the poly(A) addition site. In contrast, all three components are required for accurate cleavage and polyadenylation of pre-mRNA substrates. Further purification of CPF by buoyant density centrifugation, ion exchange, and affinity column chromatography or by gel filtration demonstrates that CPF activity resides in a ribonucleoprotein and copurifies with U11 snRNP.     

Purification and reconstitution of the high affinity choline transporter

The high-affinity choline transporter has been solubilized from synaptosomal membranes by various detergents. The solubilized carrier protein has been incorporated into liposomes after removal of the detergent by dialysis. Using the reconstitution of choline transport activity as an assay, the components catalyzing choline translocation were purified from the detergent extract by ion-exchange chromatography on a Mono-Q column followed by immunoaffinity chromatography. Monitoring the active fractions by sodium dodecylsulfate polyacrylamide gel electrophoresis and isoelectrofocussing gave one major protein with an apparent molecular weight of about 90,000 and an isoelectric point of pH 4.7. The isolated protein appeared to be heavily glycosylated as shown by lectin binding; upon treatment with endoglycosidase F the polypeptide was degraded to an apparent molecular weight of about 65,000. Accumulation of choline into liposomes reconstituted with the purified protein was driven by artificially imposed sodium gradients and inhibited by hemicholinium-3.     

Isolation and properties of the alpha-latrotoxin receptor.

The receptor protein of alpha-latrotoxin (alpha LTx, a neurotoxin with 'pure' presynaptic action isolated from black widow spider venom), was solubilized by Triton X-100 from bovine brain membranes and purified by affinity chromatography on alpha LTx-Sepharose. The purified receptor preparation contained four major polypeptides of molecular masses 200 (alpha), 160 (alpha'), 79 (beta) and 43 (gamma) kd according to SDS electrophoresis with molecular ratio alpha 1 alpha' 1 beta 2 gamma 2. The alpha- and alpha'-subunits are glycoproteins binding to wheat germ lectin and can be separated under non-denaturing conditions by anion exchange chromatography. Purified to homogeneity, both of them, though differing in the carbohydrate composition, retain the alpha LTx-binding activity and give closely related peptide maps. Anti-alpha antibodies recognize the alpha'-subunit as well. These results suggest that alpha LTx receptor is present in purified preparations in two very close forms containing the alpha- or alpha'-subunit. Beta and gamma proteins do not specifically bind alpha LTx and their physiological role is unclear. They form a complex with solubilized alpha- and alpha'-subunits independently of alpha LTx presence. The receptor proteins were purified to homogeneity by high performance gel filtration in the presence of SDS, their amino acid composition was determined.     

Adrenal Medullary Tropomyosins: Purification and Biochemical Characterization

Tropomyosins have been isolated from bovine adrenal medulla. Purified from a heat-stable extract, the adrenal medullary tropomyosins show the same chromatographic patterns as platelet tropomyosin components purified under very similar conditions on ion-exchange (DEAE-Sephacel) and hydroxylapatite columns. When analyzed by polyacrylamide gel electrophoresis, the purified fraction, reduced and denatured, yielded three polypeptides with apparent molecular weights of 38,000, 35,500, and 32,000. The molar ratio of the two major polypeptides (38 kd and 32 kd) was 2:1. The predominant form of 38 kd is different from other nonmuscle tropomyosins previously isolated and with which an apparent molecular weight of 30,000 is normally associated. The three adrenal medullary tropomyosins have similar isoelectric points of about 4.7. When adrenal tropomyosins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 8 M urea, each form showed a shift to a higher molecular weight, which is a characteristic of muscle tropomyosin. The 38,000 adrenal medullary tropomyosin exhibits a stronger affinity for F-actin than the other forms. Peptide profiles obtained after limited proteolytic digestion show some similarity between the two predominant tropomyosins of the bovine adrenal medulla and also between these and the alpha and beta forms of bovine skeletal muscle tropomyosin.     

Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.     

Regulatory subunit of cyclic AMP-dependent protein kinase I from porcine skeletal muscle: purification and proteolysis.

The regulatory subunit of cyclic AMP-dependent protein kinase I was purified to homogeneity from porcine skeletal muscle by two different procedures, one relying on affinity chromatography with cyclic AMP-Sepharose and the other relying exclusively on ion-exchange and molecular seive chromatography. Both procedures were adapted so that catalytic subunit also could be purified from the same enzyme preparation. In its native form the regulatory subunit was a dimer having a molecular weight of 92,500. Polyacrylamide gels run under denaturing conditions indicated that the dimer was composed of two identical subunits having a molecular weight 45,500. In addition to the dimeric regulatory subunit, a second, smaller cyclic AMP-binding protein frequently was observed. This protein having a molecular weight of 34,500 also was purified to homogeneity and appeared to be a proteolytic fragment derived from the regulatory subunit. Limited proteolysis with trypsin converted the regulatory subunit into a protein having a molecular weight of 34,500 and a polypeptide fragment having a molecular weight of approximately 11,000. Although the 34,500 molecular weight protein retained its capacity to bind cyclic AMP, it was monomeric apparently having lost its ability to aggregate to a dimer.     

Separation of 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities from clostridium absonum ATCC# 27555 and cellular response of this organism to bile acid inducers

Both 7 alpha- and 7 beta-hydroxysteroid dehydrogenases (HSDH) were induced by either chenodeoxy-(CDC) or deoxycholic (DC) acid in C. absonum. 7 beta-HSDH was partially purified 35-fold from CDC-induced cultures of C. absonum by Procion Red (PR) affinity chromatography and high performance liquid chromatography (HPLC) using a TSK 3000 SW gel filtration column. A relative molecular weight of 200 K was estimated for 7 beta-HSDH using Sephacryl S-300 chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the 35-fold purified 7 beta-HSDH showed six polypeptides in the molecular weight range of 40-50 K. Induction of cultures of C. absonum with CDC or DC (0.4 mM) also resulted in the differential synthesis of at least five new polypeptides with molecular weights of 94 K, 42 K, 32 K, 21 K, and 16 K. The 16 K polypeptide was induced by DC but not by CDC. SDS-PAGE of Triton X-100-solubilized membranes from these extracts revealed the presence of a new membrane-associated polypeptide of molecular weight 80 K. The soluble inducible polypeptides were eliminated during purification of the 7 alpha- and 7 beta-HSDH and, therefore, are not required for these enzyme activities. It is proposed that this organism synthesized 7 alpha- and 7 beta-HSDH as well as a series of other proteins in response to bile acids which may, in the absence of the dehydrogenases, be toxic to C. absonum. The HSDH's catalyze the epimerization of chenodeoxycholic acid to ursodeoxycholic acid, which is less toxic than the chenodeoxycholic acid. The other proteins may assist the survival of the organism in a high bile acid environment by mechanisms not yet understood.     

Baculovirus-Mediated Large-Scale Expression and Purification of a Polyhistidine-Tagged Rubella Virus Capsid Protein

The capsid protein of rubella virus was produced in baculovirus-infectedSpodoptera frugiperdainsect cells, with a polyhistidine affinity tag at the carboxy terminus. The RV capsid recombinant protein was produced in a 10-liter bioreactor and purified, under nondenaturing conditions, using immobilized metal–ion affinity chromatography. Immunoblot analyses indicated that the purified recombinant protein was intact and migrated with the expected molecular weight. The final yield was 5 mg of purified protein per liter of cell culture. Surface plasmon resonance was used to investigate the antigenic potential of the histidine tagged capsid protein in an antigen–antibody interaction study. A specific interaction between the two proteins was shown. Our results suggest that this strategy should be useful in interaction studies of other virus-specific proteins and antibodies.     

Plasmodium falciparum Rhoptry Proteins of 140/130/110 kd (Rhop-H) Are Located in an Electron Lucent Compartment in the Neck of the Rhoptries

ABSTRACT. To investigate in more detail the structure of the high molecular weight rhoptry protein complex of Plasmodium falciparum, Rhop-H (140/130/110 kd), the complex was affinity purified from parasite extracts using rhoptry protein specific antisera prepared against Rhop-H proteins bound to and eluted from Balb/c mouse erythrocytes, using 0.5 M NaCl. The individual proteins (140 kd/Rhop-1, 130 kd/Rhop-2, and 110 kd/Rhop-3) were separated, electroeluted, and monospecific polyclonal antisera prepared against the individual proteins, and against the affinity purified complex. Immunofluorescence assays and immunoelectron microscopic studies were performed to verify the subcellular localization of the Rhop-H epitopes. Immunoblotting and immunoprecipitation assays were also performed. We report novel findings regarding the localization of the rhoptry proteins to an electron lucent compartment in the neck of the rhoptries. Analysis of the amino acid composition of the individually purified Rhop-H proteins demonstrated a predominance of negatively charged (E, D) as well as hydrophobic residues (L, A, P, S) in the three proteins. The percentage of negatively charged residues was high for all three proteins. Similarities in amino acid composition for the three proteins supports the previous data demonstrating shared properties such as erythrocyte and liposome binding, for the three proteins. Results of antibody characterizations using rhoptry protein specific antisera demonstrate the immunodominance of the Rhop-H complex.     

The purified colicin S8 is a multimeric protein.

Bacteriocins have been isolated both as simple proteins and as proteins in association with carbohydrates, lipids, etc. Colicins are commonly inducible and extracellular. Their molecular masses range from 30 to 90 kDa. Pure colicin S8 was obtained in three steps from supernatant of induced cells: (i) Ammonium sulfate precipitation; (ii) anion exchange chromatography; and (iii) phenyl-Sepharose hydrophobic chromatography, either by preparative or fast performance liquid chromatography (FPLC) analytical purification procedure. In our hands, purified colicin S8 was an aggregation of extremely related polypeptides. Composition of those active fractions was the same: five polypeptides of molecular weight around 55 kDa. Behavior on molecular filtration indicated a molecular weight higher than 200 kDa. Similar results were obtained when purification was carried out through FPLC. Producing strains contain a single plasmid that encodes colicin S8; in minicells, this plasmid was shown to specify a 60 kDa polypeptide. We conclude that more than one form of colicin S8 exists. The forms are structurally related and can be recognized by antibodies raised against one of the polypeptides. Consistent with this conclusion, comparison of peptides produced after hydrolysis with chlorosuccinamide indicated that the active proteins contained both shared and unique components.     

Monoclonal antibody identifies a 200-kDa subunit of the dihydropyridine-sensitive calcium channel

A monoclonal antibody, mAb 1A, that immunoprecipitates the [3H]PN200-110-binding complex from rabbit skeletal muscle has been used to study the subunit structure of the dihydropyridine-sensitive, voltage-activated calcium channel. Digitonin-solubilized [3H]PN200-110-binding component, purified by wheat germ agglutinin chromatography, sediments as a 21 S complex. The sedimentation coefficient of the complex is increased to about 24 S after incubation with mAb 1A IgG. Four polypeptides with apparent molecular weights under nonreducing conditions of 220,000, 200,000, 61,000, and 33,000 co-sediment with the 21 S complex. mAb 1A recognizes the Mr 200,000 polypeptide, as shown by Western blotting analysis. [3H] PN200-110 complex purified by wheat germ agglutinin chromatography followed by immunoaffinity chromatography on an mAb 1A column is comprised primarily of the same four polypeptides. When analyzed by sodium dodecyl sulfate gel electrophoresis under reducing conditions, the Mr 220,000 protein migrates as a polypeptide of Mr 143,000; the mobility of the Mr 200,000 protein recognized by mAb 1A is unaffected by reduction. Thus, the Mr 200,000 polypeptide appears to be a previously undescribed component of the dihydropyridine-binding complex and, in association with the other polypeptides, may comprise the voltage-sensitive calcium channel.     

Resolution and properties of a protein kinase catalyzing the phosphorylation of a wheat germ cytokinin-binding protein

The major cytokinin binding protein of wheat germ (CBP) was extensively purified employing chromatography on Cibacron F3GA-Sepharose CL6B and concanavalin A-agarose as key purification steps. The major polypeptides present in the purified CBP preparations have molecular weights of 60,000 ± 4,000, 42,000 ± 3,000, and 37,000 ± 3,000, respectively. A protein kinase that catalyzes the phosphorylation of CBP (CBP kinase) was extensively purified from wheat germ by affinity chromatography on casein-Sepharose 4B and CBP-Sepharose 4B. The purification procedure resolves CBP kinase from an abundant casein kinase that does not phosphorylate CBP. CBP kinase catalyzes the phosphorylation of casein, phosvitin, CBP, and the wheat germ cyclic AMP-binding protein cABPII. CBP kinase phosphorylates the major 60,000 dalton subunit of CBP as well as 16,000 to 18,000 dalton polypeptides present in CBP preparations. CBP fractions with differing activities as substrates for CBP kinase were partly resolved by gel filtration and by chromatography on DEAE-Sephacel.     

Purification and characterization of an early glycoprotein from adenovirus type 2-infected cells.

An adenovirus type 2 early glycoprotein with an apparent molecular weight of 19,000 (E19K) in sodium dodecyl sulfate-polyacrylamide gels has been extensively purified. Purification involved detergent solubilization of membrane fractions from infected cells, followed by affinity chromatography on a lectin column and DEAE-Sephadex chromatography. The purified material contained three polypeptides (E40K, E19K, E17.5K), with approximately 90% of the material in the E19K moiety. All three polypeptides yielded identical tryptic peptide maps. The E19K polypeptide contained glucosamine as revealed by [3H]glucosamine labeling of infected cells and amino acid analysis of the purified protein. Immunoprecipitation with a monospecific antiserum showed that the E19K polypeptide started to be synthesized at 2 h, with a maximal rate at 4 h after infection. It was also synthesized at a low rate late in the infectious cycle (12 to 24 h postinfection). Immunoprecipitation from three adenovirus type 2-transformed hamster embryo cell lines and two adenovirus type 2-transformed rat cell lines revealed that one of the hamster cell lines (ad2HE4) and one of the rat cell lines (A2T2C4) expressed this protein.