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R F Tucker

Publications and source records attributed to R F Tucker.

11 recordsLinked to original sources
PubMed1994

Crystallization and preliminary crystallographic data of recombinant human osteogenic protein-1 (hOP-1).

We have obtained trigonal crystals of recombinant human osteogenic protein-1 (hOP-1), a member of the transforming growth factor-beta (TGF-beta) superfamily. hOP-1 (also referred to as BMP-7) is a bone morphogenetic protein and is active as a dimer of M(r) 32 to 36 kDa. The crystals have the symmetry of space group P3(1)21 or the enantiomorph P3(2)21 with unit cell dimensions of a = b = 99.46 A, c = 42.09 A. The crystals diffract to 2.2 A resolution and there is one hOP-1 monomer per asymmetric unit. In this paper we describe the first crystallization of a bone morphogenetic protein and present the results of preliminary X-ray diffraction data from the native protein and two heavy-atom derivatives.

Bone Morphogenetic Protein 7↗
PubMed1994

Osteogenic protein-1 (OP-1) expression and processing in Chinese hamster ovary cells: isolation of a soluble complex containing the mature and pro-domains of OP-1.

We have characterized the expression and processing of Osteogenic Protein-1 (hOP-1), a bone morphogenic protein of the TGF-beta family, in Chinese hamster ovary cells. The hOP-1 is initially synthesized as a monomeric 50 kDa pro-protein that is dimerized, glycosylated, and then proteolytically cleaved at the Arg-Xaa-Xaa-Arg maturation site in an acidic cellular compartment before secretion into the medium. Of the four potential N-linked glycosylation sites two are used, one in the mature domain and one in the pro-domain. Gel permeation chromatography of secreted hOP-1 in physiological buffers yields an apparent molecular weight of 110-120 k, indicating that after proteolytic processing the two pro-domains remain non-covalently associated with the disulfide linked mature dimer in a complex termed soluble hOP-1. Purified soluble hOP-1 is significantly more soluble in physiological buffers than the purified mature OP-1.

Amino Acid Sequence↗
PubMed1993

Drosophila transforming growth factor beta superfamily proteins induce endochondral bone formation in mammals.

Both decapentaplegic (dpp) protein and 60A protein have been implicated in pattern formation during Drosophila melanogaster embryogenesis. Within the C-terminal domain, dpp and 60A are similar to human bone morphogenetic protein 2 (75% identity) and human osteogenic protein 1 (70% identity), respectively. Both recombinant human bone morphogenetic protein 2 and recombinant human osteogenic protein 1 have been shown to induce bone formation in vivo and to restore large diaphyseal segmental defects in various animal models. We examined whether the Drosophila proteins, dpp and 60A, have the capacity to induce bone formation in mammals by using the rat subcutaneous bone induction model. Highly purified recombinant dpp and 60A induced the formation of cartilage, bone, and bone marrow in mammals, as determined by histological observations and by measurements of the specific activity of alkaline phosphatase and calcium content of the implants, thereby demonstrating that related proteins from phylogenetically distant species are capable of inducing bone formation in mammals when placed in sites where progenitor cells are available.

Amino Acid Sequence↗
PubMed1992

Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro.

We reported previously that a 32-36-kDa osteogenic protein purified from bovine bone matrix is composed of dimers of two members of the transforming growth factor (TGF)-beta superfamily: the bovine equivalent of human osteogenic protein-1 (OP-1) and bone morphogenetic protein-2a, BMP-2a (BMP-2). In the present study, we produced the recombinant human OP-1 (hOP-1) in mammalian cells as a processed mature disulfide-linked homodimer with an apparent molecular weight of 36,000. Examination of hOP-1 in the rat subcutaneous bone induction model demonstrated that hOP-1 was capable of inducing new bone formation with a specific activity comparable with that exhibited by highly purified bovine osteogenic protein preparations. The half-maximal bone-inducing activity of hOP-1 in combination with a rat collagen matrix preparation was 50-100 ng/25 mg of matrix as determined by the calcium content of day 12 implants. Evaluation of hOP-1 effects on cell growth and collagen synthesis in rat osteoblast-enriched bone cell cultures showed that both cell proliferation and collagen synthesis were stimulated in a dose-dependent manner and increased 3-fold in response to 40 ng of hOP-1/ml. Examination of the expression of markers characteristic of the osteoblast phenotype showed that hOP-1 specifically stimulated the induction of alkaline phosphatase (4-fold increase at 40 ng of hOP-1/ml), parathyroid hormone-mediated intracellular cAMP production (4-fold increase at 40 ng of hOP-1/ml), and osteocalcin synthesis (5-fold increase at 25 ng of hOP-1/ml). In long-term (11-17 day) cultures of osteoblasts in the presence of beta-glycerophosphate and L(+)-ascorbate, hOP-1 markedly increased the rate of mineralization as measured by the number of mineral nodules per well (20-fold increase at 20 ng of hOP-1/ml). Direct comparison of TGF-beta 1 and hOP-1 in these bone cell cultures indicated that, although both hOP-1 and TGF-beta 1 promoted cell proliferation and collagen synthesis, only hOP-1 was effective in specifically stimulating markers of the osteoblast phenotype.

Alkaline Phosphatase↗
PubMed1985

Type beta transforming growth factor/growth inhibitor stimulates entry of monolayer cultures of AKR-2B cells into S phase after a prolonged prereplicative interval.

Type beta transforming growth factor/growth inhibitor (TGF-beta/GI) is demonstrated to be a potent stimulator of DNA synthesis in AKR-2B mouse embryo cells with a prolonged (greater than 24 hr) prereplicative phase when compared with other growth factors (epidermal growth factor, platelet-derived growth factor, or fibroblast growth factor) that induce DNA synthesis 12-14 hr after stimulation. In addition, TGF-beta/GI inhibits the early peak of DNA synthesis produced by EGF and insulin before the later stimulatory effects of TGF-beta/GI become manifest. TGF-beta/GI induces a marked morphologic transformation in these cells prior to their entry into S phase. Like the other growth factors, TGF-beta/GI stimulates an early increase in the rate of protein synthesis in AKR-2B cells and its stimulatory effect on DNA synthesis is enhanced by insulin. The data show that this molecule is a growth factor for certain mesenchymal cells in monolayer culture but only after a prereplicative phase that is significantly longer than that of other growth factors.

Animals↗
PubMed1984

Growth inhibitor from BSC-1 cells closely related to platelet type beta transforming growth factor.

Purified growth inhibitor from BSC-1 cells and type beta transforming growth factor from human platelets are shown to have nearly identical biological activity and to compete for binding to the same cell membrane receptor. These findings suggest that the growth inhibitor and the type beta transforming growth factor are similar molecules. The data also show that the same purified polypeptide can either stimulate or inhibit cell proliferation depending on the experimental conditions.

Animals↗
PubMed1984

Specific binding to cultured cells of 125I-labeled type beta transforming growth factor from human platelets.

Purified type beta transforming growth factor from human platelets (TGF beta) radioiodinated with 125I-labeled Bolton and Hunter reagent was found to bind to a variety of cultured cells of both epithelial and mesenchymal origin, including normal human fibroblasts and keratinocytes. TGF beta binding sites have also been found on three mouse embryo-derived fibroblast-like cell lines with lower levels of TGF beta binding on the chemically transformed derivatives of these cell lines. A variety of human tumor cell lines was shown to have an inverse correlation between their level of TGF beta binding and their ability to form colonies in soft agar. The mouse embryo-derived AKR-2B (clone 84A) cells reached maximal binding of 125I-labeled TGF beta after 2 hr at 22 degrees C. Scatchard analysis of the equilibrium binding of TGF beta to AKR-2B (clone 84A) cells gives a Kd of 33 pM with approximately equal to 10,500 binding sites per cell. This Kd for TGF beta binding to AKR-2B (clone 84A) cells agreed well with the ED50 of 40 pM for stimulation of colony formation of these cells by TGF beta. The TGF beta binding sites on the AKR-2B cells were shown to be specific for TGF beta with no significant competition with epidermal growth factor, fibroblast growth factor, or insulin and only a small level of competition with high concentrations of platelet-derived growth factor. Partially purified preparations with TGF beta-like activity from mouse embryos and medium conditioned by mouse embryo-derived cells competed effectively for binding to the TGF beta receptor.

Animals↗
PubMed1983

Comparison of intra- and extracellular transforming growth factors from nontransformed and chemically transformed mouse embryo cells.

Multiple transforming growth factors (TGFs) have been isolated from the serum-free conditioned media and acid-ethanol cell extracts of nontransformed AKR-2B and chemically transformed AKR-MCA mouse cells. The TGF activity was analyzed using Bio-Gel P-60 chromatography in 1 M acetic acid and tested for colony-stimulating activity in soft agar using AKR-2B, AKR-2B (clone 84A), NRK, and AKR-MCA cells as indicators. Both intracellular and extracellular TGF activity from AKR-MCA and AKR-2B cells show a major peak of AKR-2B and epidermal growth factor-potentiated NRK colony-stimulating activity that coelute in the 13,000 +/- 2,000 molecular weight region. In the 24,000 +/- 7,000 molecular weight range, AKR-MCA cells produce intracellular and extracellular NRK colony-stimulating activity that is not potentiated by epidermal growth factor, while the intracellular and extracellular NRK colony-stimulating activity produced by AKR-2B cells requires added epidermal growth factor for colony formation. Also important in determining the growth and morphological characteristics of a cell line, besides the production of a TGF, is the ability of a cell to respond to TGF activity. We have shown that the transformed AKR-MCA cells form more colonies than AKR-2B cells in response to certain TGF activities. This suggests that the increased responsiveness of AKR-MCA cells to TGFs may be important in determining its phenotype.

Animals↗
PubMed1982

Serum contains a platelet-derived transforming growth factor.

High concentrations of fetal bovine serum induced colony formation in soft agar by anchorage-dependent, nontransformed mouse AKR-2B and rat NRK cells. The colony-stimulating activity in fetal bovine serum was precipitated by 45% saturated ammonium sulfate and migrated in molecular sieve chromatography as a single peak of activity in the 10,000-15,000 molecular weight range. The colony-stimulating activity was heat and acid stable and was destroyed by trypsin and dithiothreitol, indicating the activity is due to a polypeptide that requires disulfide bonds for biological activity. No competition for binding to the epidermal growth factor receptor was associated with the colony-stimulating activity. Isoelectric focusing revealed activity in the pI 4-5 range. The colony-stimulating activity in serum appeared to be of platelet origin because platelet-poor plasma and platelet-poor plasma-derived serum contained little activity, whereas acid/ethanol extracts of bovine and human platelets had potent colony-stimulating activity. Chromatography of platelet extracts on Bio-Gel P-60 revealed peaks of AKR-2B colony-stimulating activity in the 12,000 and 20,000 molecular weight ranges. The other biological and chemical properties of the platelet colony-stimulating activity were the same as those for the serum activity. The data indicate the presence in serum of a platelet-derived growth factor(s) with properties similar to those of the transforming growth factors.

Animals↗
PubMed1981

Protein dye affinity chromatography using immobilized tetraiodofluorescein.

The chromatographic behavior of a heterogeneous protein mixture and of a series of homogeneous proteins on the immobilized dye tetraiodofluorescein has been observed and analyzed. Less than 6%, of the millimolar concentration of dye immobilized to a porous agarose matrix is accessible to protein. The affinity of a protein for immobilized dye is dramatically increased by insertion of apolar spacer atoms between the dye and the matrix. Dye columns constructed with a 9-atom spacer can be used to advantage for the retention and competitive elution of proteins not found previously amenable to dye chromatography.

Chromatography, Affinity↗