Crystallization of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus and preliminary characterization of two crystal forms

The light-harvesting protein phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus Cohn has been crystallized in two different crystal forms by vapour diffusion. In 5% (w/v) polyethylene glycol at pH 8.5, hexagonal crystals of space group P63 with cell constants a = b = 158 A, c = 40.6 A were obtained, which turned out to be almost isomorphous with the hexagonal crystals of C-phycocyanin from the same organism. Consequently, the conformation of both phycobiliproteins must be very similar. From 1.5 M-ammonium sulfate (pH 8.5), orthorhombic crystals of space group P2221 with cell constants a = 60.5 A, b = 105 A, c = 188 A could be grown. Density measurements of these crystals indicate that the unit cell contains 18 (alpha beta)-units. A detailed packing scheme is proposed that is consistent with the observed pseudo-hexagonal X-ray intensity pattern and with the known size and shape of (alpha beta)3-trimers of C-phycocyanin. Accordingly, disc-like (alpha beta)3-trimers are associated face-to-face and stacked one upon another in rods with a period of 60.5 A, corresponding to the cell dimension a.     

Mapping the receptor-recognition site of human transforming growth factor-alpha.

The receptor-recognition site human transforming growth factor-alpha (TGF alpha), a 50-residue tricyclic peptide with three disulfide bonds, was mapped by a set of 46 peptide analogs consisting of linear, monocyclic, bicyclic, and tricyclic structures representing individual and overlapping subdomains of human TGF alpha. Linear overlapping fragments ranging from 7 to 18 residues and spanning the entire length of TGF alpha as well as monocyclic analogs with one disulfide linkage were found to be inactive in both receptor-binding and mitogenic assays. Bicyclic analogs with two disulfide linkage and representing either the amino or carboxyl two-thirds of TGF alpha showed low activity at 0.1-0.9 mM concentrations. Tricyclic analogs containing all three disulfide linkages but lacking either the amino or carboxyl terminal heptapeptide was, respectively, 3% and 0.1% as active as TGF alpha. These results show that determinants for the receptor binding cannot be represented by a short continuous fragment or a single subdomain, but are located on a discontinuous surface on a folded structure with disulfide restraints. Furthermore, these results when combined with our previous results which shows that the middle subdomain (second disulfide loop) is not involved in the receptor binding suggest that the receptor-binding residues are constituted of three fragments located at the first and third subdomains as well as the external carboxyl peptide.     

Dioxin induces transforming growth factor-alpha in human keratinocytes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental toxicant, is a tumor promoter that induces hyperplasia in epithelial cells. Exposure of cultured human keratinocytes to TCDD, resulted in a time-dependent dioxin-specific Ah receptor-mediated release of transforming growth factor-alpha (TGF-alpha) into the culture medium. Cultures exposed to TCDD showed a rate of TGF-alpha secretion into the medium of about 30 fmol/ml/day, as well as a 3- to 6-fold increase in TGF-alpha mRNA expression. Increased production of TGF-alpha in human keratinocytes exposed to TCDD demonstrates a modulation of autocrine regulation in those cells. These results suggest that induction of TGF-alpha could be an important part of the mechanism of dioxin-mediated toxicity and tumor promotion.     

Human plasminogen kringle 4. Crystallization and preliminary diffraction data of two different crystal forms

Human plasminogen kringle 4 has been crystallized in two different crystal forms: monoclinic, a = 32.78(3), b = 49.17(2), c = 46.27(3) A, beta = 100.67 degrees, space group P2(1), four molecules/unit cell, two molecules/asymmetric unit; orthorhombic, a = 32.09(7), b = 49.14(6), c = 49.47(9) A, space group P2(1)2(1)2, four molecules/unit cell. Both crystal forms have a large protein fraction (66% for monoclinic and 62% for orthorhombic) and diffract x-rays to 2.0 A resolution. A self-rotation function has been calculated with monoclinic data indicating a non-crystallographic 2-fold rotation approximately parallel to a* (peak height of 14.3 x sigma). Cross-rotation function calculations are in progress utilizing the coordinates of the conserved structure of kringle 1 of prothrombin and plasminogen kringle 4.     

Crystallization and preliminary X-ray investigation of recombinant human interleukin 4

Crystals of recombinant human interleukin 4 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space-group P4(1)2(1)2 or P4(3)2(1)2; the unit cell axes are a = 92.2(1) A and c = 46.4(1) A. The crystals are stable to X-rays for at least three days and diffract beyond 2.8 A resolution. The crystals contain approximately 63% solvent, assuming there is one molecule in the asymmetric unit.     

Crystallization and preliminary X-ray investigation of recombinant human interleukin 10

Crystals of recombinant human interleukin 10 have been grown from solutions of ammonium sulfate. The crystals are tetragonal, space group P4₁2₁2 or P4₃2₁2; the unit cell axes are a = 36.5 Å and c = 221.9 Å. There is the equivalent of one polypeptide chain in the asymmetric unit. The crystals are stable to X-rays and diffract to at least 2.5 Å resolution. © 1995 Wiley-Liss, Inc.     

Purification and characterization of three forms of differently glycosylated recombinant human granulocyte-macrophage colony-stimulating factor

We have purified recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) produced in human lymphoblastoid Namalwa cells. From the results of tunicamycin treatment and N-glycosidase F digestion, it was demonstrated that Namalwa-derived hGM-CSF was highly glycosylated at two potential N-glycosylation sites and several O-glycosylation sites as previously shown for naturally occurring hGM-CSF. We classified the hGM-CSF molecules into three groups according to the molecular weight corresponding to the degree of N-glycosylation: the molecules with two N-glycosylation sites occupied (designated 2N), the molecules with either site glycosylated (1N), and the molecules lacking N-glycosylation (0N). Despite such varied degrees of N-glycosylation, almost all molecules were O-glycosylated. To investigate the role of carbohydrate moieties of hGM-CSF, we isolated each form of hGM-CSF and examined its biological properties. The 2N-type showed 200-fold less in vitro specific activity compared with unglycosylated Escherichia coli-derived hGM-CSF, although the activity of the 0N-type was equivalent to that of the E. coli-derived material. The 1N-type showed an intermediate level of activity. However, in terms of clearance from blood circulation in the rat, the 2N-type showed a half-life five times longer than that of the 0N-type and E. coli-derived hGM-CSF. From these findings, we concluded that N-linked carbohydrate moieties of hGM-CSF play conflicting physiological roles in the efficacy of the protein in vivo but that O-linked carbohydrate moieties do not have such effects.     

Induction of transforming growth factor-alpha in activated human alveolar macrophages

The early monocyte infiltration observed in normal wound repair and in a number of pathologic processes precedes the epithelial and connective tissue proliferative responses, suggesting that the monocyte/macrophage may be an important source of growth factors for these tissues. In culture, activated macrophages secrete growth factors active on fibroblasts, smooth muscle, endothelium, and epithelium. This report demonstrates that activated human alveolar macrophages express the gene for transforming growth factor-alpha (TGF-alpha) in an inducible manner and secrete a factor into the culture medium that is functionally and immunologically identical to TGF-alpha. Two different molecular species of TGF-alpha activity (approximately 8,500-12,000 and 28,500 daltons) are identified in macrophage-conditioned medium. These observations establish the macrophage as a diploid human cell capable of synthesizing and secreting TGF-alpha. The activated macrophage therefore represents a cellular source of a mitogenic factor that is potentially important in epithelial proliferation and repair.     

Crystallization and preliminary X-ray diffraction data of two crystal forms of bovine heart creatine kinase

Two crystal forms of bovine heart creatine kinase, which are suitable for X-ray diffraction studies, have been grown at room temperature using 2-methyl-2,4-pentanediol as the precipitant at pH 7.2. The space group of the orthorhombic form is P2(1)2(1)2, with unit cell dimensions a = 133 A, b = 128 A and c = 65 A, and there is one dimeric molecule in the asymmetric unit. The space group of the tetragonal form is P4(2)2(1)2, with unit cell dimensions a = b = 132 A and c = 75 A, with one subunit in the asymmetric unit. The tetragonal crystals diffract to at least 2.0 A resolution.     

Crystallization and preliminary X-ray investigation of a recombinant form of human gamma-interferon

A genetically engineered human gamma-interferon has been crystallized from solutions of ammonium sulfate. The crystals are trigonal, space group R32, with hexagonal axes of a = 114.0(1) A and c = 314.9(2) A. The crystals are moderately stable to x-rays and diffract to 2.85-A resolution. Density measurements indicate that the crystallographic asymmetric volume contains four interferon molecules.     

Crystallization and preliminary x-ray characterization of bovine growth hormone. Purification of bovine prolactin and growth hormone

A new purification scheme for both prolactin and growth hormone from bovine pituitaries has been developed which avoids the use of potentially damaging solution conditions. Both hormones were greater than 95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had specific activities similar to or greater than standard samples of the same hormone as judged by several bioassays. Small single crystals of bovine growth hormone were obtained by vapor diffusion techniques. Examination of these crystals by x-ray diffraction, using the Cornell High Energy Synchrotron Source, showed that they were well ordered, and exhibited diffraction to 2.8-A resolution on still photographs. Precession and oscillation photographs showed that they belonged to the orthorhombic space group P2(1)2(1)2(1) (or P2(1)2(1)2) with unit cell dimensions a = 219 A, b = 51.9 A, c = 68.9 A. The density of the crystals was 1.19 +/- 0.02 g/ml from which the presence of eight 45,000-dalton dimers/unit cell was deduced. The protein content of the crystals was shown by isoelectric focusing to be identical to that of purified growth hormone in solution. These crystals appear suitable for use in the x-ray structure determination of bovine growth hormone to at least 3.2-A resolution.     

Crystallization and preliminary x-ray diffraction studies of recombinant human interleukin-1 beta

Recombinant human interleukin-1 beta has been crystallized into a tetragonal cell. The unit cell constants are a = b = 54.9 A, c = 76.8 A, and alpha = beta = gamma = 90 degrees. The crystals diffract to better than 1.9 A and are suitable for high resolution data collection. The crystallization conditions and general crystal data are presented.     

Crystallization and preliminary X-ray studies on human epidermal growth factor

Single crystals of human epidermal growth factor have been prepared from a polyethylene glycol solution and characterized by X-ray diffraction. The crystals grow in a space group of P2(1) with unit cell dimensions of a = 32.7, b = 32.5, c = 22.3 A, and beta = 96.9 degrees. They contain a single molecule per asymmetric unit and show better diffraction than 2.5 A.     

Crystallization and preliminary crystal data of porcine pepsinogen

Single crystals of porcine pepsinogen, suitable for x-ray diffraction studies, have been grown with lithium sulfate as the precipitant. These pepsinogen crystals were dissolved, activated, and assayed for proteolytic activity. The specific enzymic activity of the dissolved crystalline protein was nearly twice that of the commerical pepsinogen from which the crystals were grown. Incubation at pH 8 before assay demonstrated that the crystals are free of pepsin. This crystal form of pepsinogen belongs to the monoclinic space group C2 with 4 molecules in the unit cell. The unit cell dimensions are a = 104.8 +/- 0.5 A, b = 43.1 +/- 0.1 A, c = 88.4 +/- 0.3 A, and beta = 91.3 degrees.     

Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor-beta

Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.     

Chemical synthesis of bovine transforming growth factor-alpha: synthesis, characterization and biological activity

Bovine transforming growth factor-alpha (bTGF-alpha) is a 50 amino acid polypeptide with three disulfide linkages. In order to evaluate the biological function of this peptide, bTGF-alpha was synthesized via an automatic synthesizer and purified to homogeneity in high yield. The integrity of this synthetic peptide was confirmed by chemical analyses and bioassays. In a bovine liver radioreceptor assay, bTGF-alpha competes with radiolabeled EGF and has activity comparable to mEGF and hTGF-alpha. Compared to hEGF, bTGF-alpha elicits a greater response in a bovine mammary cell proliferation.