A detergent-activated tyrosinase from Xenopus laevis. I. Purification and partial characterization

A tyrosinase has been purified from the skin of the frog Xenopus laevis. Dihydroxyphenylalanine oxidase and tyrosine hydroxylase activities co-purify throughout the procedure. The enzyme is isolated in an inactive form, but both enzymatic activities are activated by a variety of anionic detergents. Of these, sodium dodecyl sulfate (NaDodSO4) is the most effective. The enzyme activation occurs at NaDodSO4 concentrations well below the critical micelle concentration and it remains active at concentrations as high as 30 mM (1%). Neither activity is stimulated by cationic or nonionic detergents, or a variety of other agents, including trypsin. The purified tyrosinase is a glycoprotein having a polypeptide Mr = 175,000 by NaDodSO4-polyacrylamide gel electrophoresis. This monomeric species is enzymatically active in the presence of NaDodSO4. Detergent-activated tyrosinase has a KM for dihydroxyphenylalanine of 6 X 10(-4) M and a KM for tyrosine of 4 X 10(-4) M. Both activities are inhibited by copper chelators but not by an iron chelator. Further characterization of the detergent activation of this enzyme is presented in a companion paper (Wittenberg, C., and Triplett, E. L. (1985) J. Biol. Chem. 260, 12542-12546).     

Purification, partial characterization and biological effects of the XTC mesoderm-inducing factor

The mesoderm of Xenopus laevis is formed through an inductive interaction in which a signal from the vegetal hemisphere of the blastula acts on overlying animal pole cells. We have recently reported that the Xenopus XTC cell line secretes a mesoderm-inducing factor (MIF) which may resemble the natural signal. In this paper, we describe the purification and biological effects of XTC-MIF. XTC-MIF is a hydrophobic protein with an isoelectric point of 7.8 and an apparent relative molecular mass (Mr) of 23,500. On reduction, XTC-MIF loses its biological activity and the protein dissociates into two inactive subunits with apparent Mr of about 15,000. These properties closely resemble those of transforming growth factor type beta (TGF-beta), and it is interesting that TGF-beta 2 has recently been shown to have mesoderm-inducing activity. The biological response to XTC-MIF is graded. After exposure to 0.2-1.0 ng ml-1 XTC-MIF, stage-8 animal pole explants form mesenchyme and mesothelium. At higher concentrations, up to about 5 ng ml-1, muscle is formed, occasionally with neural tissue. In response to concentrations of XTC-MIF greater than 5-10 ng ml-1, notochord and neural tissue are usually formed. The formation of notochord and neural tissue in response to XTC-MIF represents a qualitative difference between this inducing factor and the other known group of MIFs, the heparin-binding growth factors.     

Purification and characterization of Xenopus laevis topoisomerase II

We have purified to apparent homogeneity a type II DNA topoisomerase from Xenopus laevis oocyte nuclei (germinal vesicles, or GV). The most pure preparations contain a single polypeptide of 175,000 daltons as determined by SDS-gel electrophoresis. The enzyme changes the linking number of DNA circles in steps of two and reversibly knots or catenates DNA rings. No gyrase activity is detectable and ATP is required.     

A mesoderm-inducing factor from a Xenopus laevis cell line

Summary We have compared the chemical properties and biological activities of the mesoderm-inducing factor that is secreted by the Xenopus XTC cell line with the vegetalizing factor from chicken embryos. The inducing activity of the factors was tested in different concentrations on totipotent ectoderm either by implantation into early gastrulae of Triturm alpestris or by application of solutions to isolated ectoderm of early gastrulae of Xenopus laevis. Both factors have similar properties. They are not irreversibly inactivated after treatment with 6 M urea or with phenol at 60° C. Reduction with thioglycolic acid inactivates the factors completely. The inducing activity of XTC-conditioned medium decreases only slightly after treatment with 50% formic acid. The apparent molecular mass and the isoelectric point of the factors are similar. The XTC factor was partially purified by size-exclusion and reversed-phase high-pressure liquid chromatography and by isoelectric focusing. The possible relationship of these factors to transforming growth factor is discussed.Dedicated to Prof. Dr. Sulo Toivonen on the occasion of his 80th birthday     

Purification and characterization of Xenopus laevis type I topoisomerase

A topoisomerase activity was purified from mature ovaries and from nuclei of stage 6 oocytes of Xenopus laevis. From both preparations we obtained a single polypeptide chain having an estimated molecular weight of 67,000. The enzyme purified from ovaries is active in the presence of 150 mM monovalent cation, but its activity is more than 1 order of magnitude higher in the presence of 6 mM Mg2+; the enzyme purified from nuclei requires Mg2+ through all the steps of purification. Enucleated oocytes are devoid of topoisomerase activity but are able to convert the nuclear enzyme to a species active also in the presence of monovalent cations. The difference in ionic requirement between the nuclear topoisomerase and the enzyme purified from ovaries as well as the topoisomerases from other eukaryotic sources, which are most active in the presence of monovalent cations, may depend on the source of the enzyme and/or on the extraction procedure. Ovarian and nuclear topoisomerases catalyze relaxation of both negatively and positively superhelical DNA; the relaxed isomers produced in the presence of Mg2+ have a few positive superhelical turns.     

Purification and characterization of an N-acetyl-beta-D-glucosaminidase from cortical granules of Xenopus laevis eggs

The enzyme N-acetyl-beta-D-glucosaminidase was purified from the cortical granules of Xenopus laevis eggs using affinity chromatography, gel filtration, and density gradient centrifugation. The enzyme had a molecular weight of 37,000-40,000 as determined by polyacrylamide gel electrophoresis and density gradient centrifugation, had a Km for p-nitrophenyl-beta-D-N-acetyl-glucosaminide of 0.66 mM and a Ki for glucosamine of 4.3 mM. The kinetic properties of the cortical granule enzyme were similar to the enzyme isolated from jack bean. Treatment of unfertilized eggs with the enzyme isolated from cortical granules or jack bean rendered eggs unfertilizable. Loss of fertilizability was proportional to the product of time and enzyme concentration, consistent with an enzymatic mechanism being responsible for the loss of fertilizability. The amount of enzyme present in the perivitelline space was approximately the same as that which reduced fertilizability by 50% in one hour. We suggest that the action of cortical granule N-acetyl-beta-D-glucosaminidase on egg integuments may function as a block to polyspermy at fertilization.     

Purification of a calcium dependent ribonuclease from Xenopus laevis.

We have purified a Ca2+ dependent ribonuclease from the oocytes of Xenopus leavis. Two properties of this ribonuclease set it apart from other known nucleases. First, Ca2+ was required for ribonuclease activity, and Mg2+ would not substitute. Second, the enzyme specifically degraded RNA and digestion of double or single stranded DNA was not observed. Ca2+ dependent ribonuclease activity of the purified 36-kDa protein was directly observed after renaturation of the protein following electrophoresis in an SDS-Laemmli gel. In addition, the enzyme was shown to have endoribonuclease activity at numerous sites. The Ca2+ dependence suggests that the ribonuclease activity may be modulated by changes in the level of intracellular Ca2+ and thereby provide a direct link to signal transduction systems.     

Isolation and characterization of calmodulin genes from Xenopus laevis.

Two cDNAs derived from Xenopus laevis calmodulin mRNA have been cloned. Both cDNAs contain the complete protein-coding region and various lengths of untranslated segments. The two cDNAs encode an identical protein but differ from each other by 5% nucleotide substitutions. The 5' and 3' untranslated regions, to the extent available, are highly homologous between the two cDNAs. The predicted sequence of X. laevis calmodulin is identical to that of vertebrate calmodulins from mammals and chickens and shows one substitution compared with electric eel calmodulin. Genomic DNA sequences homologous to each of the two cDNA clones have been isolated and were shown to account for the major calmodulin-coding DNA sequences in X. laevis. These data suggest that X. laevis carries two active, nonallelic calmodulin genes. Although no complete analysis has been carried out, it appears that the X. laevis calmodulin genes are interrupted by at least four introns. The relative concentrations of calmodulin mRNA have been estimated in different embryonic stages and adult tissues and found to vary by up to a factor of 10. The highest levels of calmodulin mRNA were found in ovaries, testes, and brains. In these three tissues, the two calmodulin genes appear to be expressed at approximately equal levels.     

Purification and characterization of a casein-kinase-II-type enzyme from Xenopus laevis ovary. Biological effects on the meiotic cell division of full-grown oocyte

A casein kinase of type II has been highly purified from Xenopus laevis ovary. A new experimental protocol has been developed for the purification, consisting in four chromatographic steps: hydrophobic on tyrosine-agarose, ion exchange on DEAE-Sepharose, affinity on heparin-Sepharose and fast protein liquid on Mono Q. The purification was greater than 20,000, taking into account an inhibitor present in the starting material which masked the activity in the crude fraction. The overall yield was greater than 20%. Full-grown Xenopus oocytes contain 64 milliunits per oocyte corresponding to an intracellular concentration in the nanomolar range. The enzyme shares the following features with the mammalian casein kinase II: (a) comparable subunit composition (42-kDa doublet, 38 kDa and 26 kDa), (b) autophosphorylation of the 26-kDa subunit, (c) ability to use GTP as well as ATP as phosphate donor, (d) inability to use Mn2+ instead of Mg2+ to support the activity, (e) phosphorylation of both threonine and serine residues of casein, (f) inhibition by low doses of heparin. Biological effects of the highly purified enzyme have been investigated upon microinjection into Xenopus full-grown oocytes. At nanomolar concentrations (approximately 3 nM) the enzyme inhibited progesterone induction of meiotic cell division whereas it facilitates meiotic maturation at the level of maturation-promoting factor. These results suggest a role for the kinase in the phosphorylation cascade involved during the prophase/metaphase transition of meiotic cell division, both in the mechanism of the meiotic prophase arrest and in the activity of the cytoplasmic factor maturation-promoting factor. When microinjected into oocytes above 45 nM, the kinase provoked complex changes in the profile of the in ovo 32P-labelled proteins indicating that its targets could be other kinase/phosphatase regulatory proteins.     

Effects of cell heterogeneity on production of polypeptide growth factors and mesoderm-inducing activity by Xenopus laevis XTC cells

The Xenopus laevis XTC cell line has been analyzed for the production of polypeptide growth factors and mesoderm-inducing activity. By the use of specific biological assays, it is shown that XTC cells produce a growth factor functionally related to the platelet-derived growth factor (PDGF) and two transforming growth factor (TGF) beta-like activities. Mesoderm-inducing activity, as measured on X. laevis ectodermal explants from stage 10 embryos, was found to coelute on a Bio-Gel P-100 column with one of the TGF beta-like activities at an apparent molecular weight of 6-10 kDa. Analysis of the DNA content from XTC cells by flow cytometry demonstrated that the cell line is heterogeneous and consists of both tetraploid and diploid cells. Cloning of the XTC cells and selecting single-cell colonies on the basis of their ability to grow in soft agar resulted in the isolation of several homogeneous, morphologically different clonal derivatives. Analysis of conditioned medium from these clonal derivatives showed that only one of them, the only diploid line among six investigated, produced a strong heat- and acid-stable mesoderm-inducing activity that induced notochord and muscle formation in stage 10 X. laevis ectodermal explants. The relation between this activity and a recently described TGF beta-like mesoderm-inducing factor obtained from XTC-conditioned medium will be discussed. In conclusion, a clonal cell line derived from X. laevis XTC cells which provides a good source for further characterization of mesoderm-inducing factors has been established.     

Purification, immunological and biochemical characterization of Ap4A binding protein from Xenopus laevis oocytes.

Diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) binding protein specifically binds Ap4A. The protein has been purified from Xenopus laevis oocytes and presents an estimated molecular weight of 100,000 by gel filtration. In the first stages of the purification, the Ap4A binding activity is found associated to DNA polymerase alpha-DNA primase, forming heterogeneous high molecular weight complexes. A monoclonal antibody has been prepared against the purified Ap4A binding protein. The antibody partially neutralizes the Ap4A binding activity. Using the immunoblot technique, it has been shown that the antibody is able to recognize either native or SDS-denatured Ap4A binding protein. The monoclonal antibody immunoreacted with a polypeptide of 90,000 which coincides with the molecular weight obtained by gel chromatography and indicates that the native Ap4A binding protein from Xenopus oocytes is probably a monomeric protein.     

Purification of a DNA-binding protein from Xenopus laevis unfertilized eggs.

A DNA-binding protein from Xenopus laevis unfertilized eggs has been purified to apparent homogeneity. It is a heat stable, lysine-rich protein and has a molecular weight corresponding to 8,200 daltons, measured by sodium dodecyl sulphate gel electrophoresis. The protein, which is active in a monomeric form, stimulates DNA polymerase alpha, and binds to single and double stranded DNA. One egg contains about 4 x 10(12) molecules (minimum estimate) of the protein; since we calculate that 4 x 10(8) molecules are sufficient to cover the entire genome (haploid complement), there is much more protein than is needed to cover chromosomal DNA.     

A mucus-secreting human colonic cancer cell line. Purification and partial characterization of the secreted mucins.

A stably differentiated clonal derivative (Cl.16E) of the human colonic adenocarcinoma cell line HT29 secretes in culture high-Mr glycoproteins that were purified from the serum-free conditioned medium by preparative SDS/polyacrylamide-gel electrophoresis. Analysis of the oligosaccharides released from the [3H]glucosamine-labelled high-Mr glycoproteins by alkaline-borohydride treatment showed that this material consisted of O-linked oligosaccharides (without any detectable N-linked oligosaccharides) that were eluted as three fractions from Bio-Gel P-6 columns. The main oligosaccharide fraction obtained after such treatment and desialylation was eluted together with a six-unit glucose polymer from a Bio-Gel P-4 column. Polyclonal antibodies were raised against the high-Mr glycoproteins, and in immunoblot analysis they reacted specifically with the high-Mr glycoproteins present in the conditioned medium. Furthermore, immunohistochemical staining of sections in paraffin wax revealed that these antibodies labelled normal human gastrointestinal mucins. We conclude that (1) the high-Mr glycoproteins prepared by SDS/polyacrylamide-gel electrophoresis are pure mucus glycoproteins on the basis of sensitivity to alkaline-borohydride treatment, monosaccharide composition and immunochemical and immunohistological findings, and (2) these mucins have antigenic determinants in common with the normal human gastrointestinal mucins.     

Purification and partial characterization of high-molecular-weight forms of ectopic calcitonin from a human bronchial carcinoma cell line.

Studies on the high-molecular-weight immunoreactive calcitonin produced ectopically in culture by an epidermoid bronchial carcinoma cell line are reported. In cell-exposed medium, the principal component has a molecular weight of 40000 and molecules of mol.wts. 13000 and 10000 also occur. Only a trace amount of material co-eluting with 35000-mol.wt. human calcitonin is detectable. None of the calcitonins show cross-reactivity with anti-corticotropin serum. The 40000-mol.wt. immunoreactive calcitonin is readily proteolysed to the 13000- and 10000-mol.wt. components, but the 10000-mol.wt. component behaves as a comparatively stable 'core' molecule. By using immunoprecipitation and high-pressure liquid chromatography (h.p.l.c.), it is possible to prepare radiochemically homogeneous 10000-mol.wt. immunoreactive calcitonin from cells grown in the presence of individual 35S- or 3H-labelled amino acids. Peptide mapping of enzymic digests of this material by h.p.l.c. shows that it contains peptides in common with synthetic human calcitonin.     

Isolation and characterization of a monosialosylgangliopentaosyl ceramide from Xenopus laevis oocyte.

A monosialosylgangliopentaosyl ceramide was isolated from Xenopus laevis oocytes. It represented 5.8% of the total acidic glycosphingolipids. From the results of sugar-composition analysis, enzymatic hydrolysis, permethylation analysis, and negative ion fast atom bombardment mass spectrometry, the structure of the ganglioside was determined to be as follows: [sequence: see text] The predominant species of fatty acids were alpha-hydroxy fatty acids, h22:0, h24:0, and h24:1. The long chain bases of this ganglioside consisted mainly of d18:1 sphingosine and phytosphingosine. Other acidic glycolipids were also characterized. The most abundant component of acidic glycolipids was sulfatide, which represented 85.7% of the total acidic glycolipid mixture. GM3, GM2, GM1a, and GD1a were also detected.     

Purification of Xenopus laevis embryo and liver nuclei using metrizamide.

While the nuclei of many diverse types of tissue can be purified by centrifugation through dense sucrose solutions, Xenopus laevis embryo and liver nuclei present special purification problems due to the presence of large numbers of melanosomes in embryo and liver cells. These melanosomes were removed by isopycnic centrifugation in Metrizamide gradients. In Metrizamide, embryo nuclei banded at an average buoyant density of ρ_5°C = 1.288 ± 0.003 g/cm³. Liver nuclei separated into two peaks, one containing nonparenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.274 ± 0.003 g/cm³ and the other peak containing parenchymal cell nuclei with an average buoyant density of ρ_5°C = 1.284 ± 0.001 g/cm³.     

Purification and partial characterization of virus-like particles from Schneider line 2 Drosophila cells

A procedure has been developed for the purification of virus-like particles (VLPs) from Schneider line 2 Drosophila cells. The VLPs were precipitated with polyethylene glycol from the cytoplasmic fraction of lysed cells and further purified by equilibrium centrifugation in CsCl density gradients, in which they band at a density of 1.366 g/ml. Electron micrographs of these preparations revealed polyhedral particles with a diameter of 310–330 Å. We have also found particles of this size in thin sections of the intact cells. Sedimentation of the VLPs through 10–70% sucrose gradients yields a sedimentation coefficient of 235 S. Preliminary studies show that the VLPs contain double-stranded RNA species of 10 S, 14.5 S, 16 S, and 18 S.