Purification and biochemical characterization of a human autocrine growth factor produced by Epstein-Barr virus-transformed B cells

Autocrine growth factor for Epstein-Barr virus-transformed human B cells (aBGF), a protein that is constitutively produced by the human EBV-transformed B cell line 5/2, has been purified from serum-free conditioned medium. The purification involved sequential ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and reversed-phase high performance liquid chromatography. The purified protein has a m.w. of 16,000 in NaDodSO4/polyacrylamide gel electrophoresis and an isoelectric point between 7.0 and 8.0. The relative molecular mass 16,000 form exists in equilibrium with dimeric and tetrameric forms. aBGF supports the growth of EBV-transformed B cells, which have been deprived of their own conditioned medium. The purified aBGF is fully effective at 0.5 ng/ml and has no interleukin 1 activity in the lymphocyte activation factor assay. Because several randomly selected lines of EBV-transformed cells and one EBV-negative lymphoma cell line both produce aBGF activity and show growth dependency on aBGF and because stimulation of normal B cells with anti-immunoglobulin M is increased by aBGF, we propose that aBGF has general significance for growth control of human B cells.     

Partial purification of murine B cell stimulatory factor (BSF)-1

BSF-1 was partially purified from serum-free culture supernatants of cells of the EL-4 thymoma line, which had been induced 48 hr earlier with 4 beta-phorbol-12 beta-myristate-12 alpha-acetate (PMA). BSF-1 in 10-liter batches was adsorbed onto and eluted from trimethylsilyl-controlled pore glass beads (TMS-CpG) and then subjected to reverse-phase high-performance liquid chromatography (RP-HPLC). The recovery of BSF-1 activity by TMS-CpG and RP-HPLC ranged from 52 to 55% and 187 to 227%, respectively. The specific activity in units per milligram of protein of partially purified BSF-1 was approximately 2600 times higher than that of the culture supernatant protein. The partially purified BSF-1 had a single isoelectric point of 6.3 and an apparent m.w. between 18,000 and 21,700 when analyzed by isoelectric focusing and gel filtration-HPLC, respectively. The ability to prepare large amounts of partially purified BSF-1 by a rapid and efficient procedure should be of great help in both biochemical and immunologic studies of this lymphokine.     

Lymphokine-activated and natural killer cell activity in human intestinal mucosa

The activity of natural effector (NE) cells was studied in lamina propria lymphocytes (LPL) obtained from 61 histologically normal specimens of human intestine, which included 45 resected for colon carcinoma and 16 resected for nonmalignant conditions. The mean spontaneous natural killer (NK) cell activity in LPL (1.7 X 10(2) cytotoxic units (C.U.)/10(5) cells) was very low in contrast to that found in peripheral blood mononuclear cells (PBMC) (38.5 X 10(2) C.U./10(5) cells). Significant NK activity was detected in only 16 (47%) of the tissues resected for carcinoma, and in five (38%) of those removed for nonmalignant conditions. Exposure to human leucocyte interferon resulted in only minimal increases in cytotoxicity for K562 target cells. Consistent with these findings, large granular lymphocytes represented less than 0.5% of freshly isolated LPL. Cultures of LPL from both carcinoma and nonmalignant conditions in MLA144-conditioned medium (CM), a source of interleukin 2 (IL 2), generated marked increases in cytotoxicity to levels comparable with or exceeding those found in PBMC. (Mean cytotoxicities were 90.4 X 10(2) and 49 X 10(2) C.U./10(5) cells, respectively.) Cytotoxicity induced by culture in MLA144-CM could be blocked by pretreatment of LPL with the monoclonal antibody anti-Tac directed against the IL 2 receptor. In addition, LPL cultured in recombinant human IL 2 were induced to levels of cytotoxicity that were similar to those induced by MLA144-CM. These data indicate that IL 2 is the factor in MLA144-CM responsible for generating lymphokine-activated killer (LAK) cells in LPL. The IL 2-activated LPL killer cells were OKT11+, OKT3-, Leu-7-, Leu-11b-, as determined by antibody and complement-mediated lysis, and the precursor cells in the lamina propria necessary for generation of killer cells by IL 2 were also OKT11+, OKT3-, Leu-7-, Leu-11b-. These studies indicate that LAK cells may be an important potential source of nonspecific cytotoxicity in the intestinal mucosa.     

Purification and characterization of a gelatinase produced by fibroblasts from human gingiva

An endopeptidase which digests denatured collagen to small, dialysable fragments was purified 2675-fold from medium that had been conditioned by the culture of fibroblasts grown from explants of human gingiva. This enzyme was inhibited by chelating agents, but not by phenylmethylsulphonyl fluoride nor by N-ethylmaleimide, and is therefore probably a metalloproteinase. It showed no demonstrable activity against native collagen or ovalbumin, while alpha-casein was digested slowly, if at all. It therefore belongs to the group of enzymes which have been called tissue gelatinases. This gelatinase was secreted in a latent form or forms and could be activated by proteolysis with trypsin. The active enzyme had an apparent molecular weight of 69 000 (gel chromatography) or 72 000 (gel electrophoresis in sodium dodecyl sulphate) and an apparent isoelectric point of 4.15.     

Purification of a colony-stimulating factor from cultured pancreatic carcinoma cells.

Serum-free conditioned medium prepared from an established line of human pancreatic carcinoma (MIA PaCa-2) provides a rich source of colony-stimulating factor (CSF). Two activities distinctly separable by isoelectrofocusing have been identified: a high molecular weight CSF exhibiting greater activity in mouse bone marrow and a low molecular weight CSF more active in human bone marrow. The high molecular weight CSF has been purified 1000-fold to apparent homogeneity by a two-step procedure including isoelectrofocusing and gel filtration chromatography. The purified CSF has a molecular weight of 50,000 and an isoelectric point of 3.7 to 4.6. It is a glycoprotein as shown by periodic acid-Schiff stain and exhibits greater activity in mouse marrow than in human marrow.     

Partial purification and characterization of a neutral proteinase with collagen telopeptidase activity produced by human gingival fibroblasts

A neutral proteinase was purified 1930-fold from medium conditioned by the culture of human gingival fibroblasts that had been stimulated to secrete enzymes by concanavalin A. This enzyme had an apparent molecular weight of 35,000 (gel chromatography) and apparent isoelectric point of 4.3 (chromatofocusing). It was inhibited by chelating agents, serum, and nonactivated conditioned fibroblast medium, but not by phenylmethylsulphonyl fluoride or N-ethylmaleimide. This proteinase removes the C-telopeptide from the alpha 1 chain of type I collagen, an activity which could be important in the degradation of collagen in the extracellular matrix. It was also found to digest fibronectin but had no effect on proteodermatan sulphate under the conditions used. It appears to be unrelated to previously described fibroblast extracellular proteinases and we, therefore, tentatively propose the name fibroblast metalloproteinase IV.     

Multiple molecular forms of purified human erythrocyte acetylcholinesterase.

1. Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein. The yield of the purified enzyme was 25--45%. 2. Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 8.7 nm. Density gradient centrifugation in 0.1% Triton X-100 showed one peak of enzyme activity with an S4 value of 6.3S. 3. Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 4.55, 4.68, 4.81, 4.98 and 5.18. Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point. 4. After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity. Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 6.3 S, 10.2 S, 12.2 S, 14.2 S and 16.3 S. At low ionic strength, higher aggregates were observed in addition to the other forms. Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80 000. 5. These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions. After removal of the detergent, forms with different molecular sizes are observed.     

Characterization of helper factors distinct from interleukin 2 necessary for the generation of allospecific cytolytic T lymphocytes

The in vitro generation of primary murine allospecific cytolytic T lymphocytes (CTL) from BALB/c (H-2d) spleen cell precursors in response to x-irradiated RDM4 (H-2k) tumor cells did not occur unless the cultures were supplemented with exogenous helper factors. Such CTL helper factors (CHF) could be provided by conditioned medium from cultures in which Sendai virus-immune BALB/c spleen cells were stimulated either with Sendai-infected cells (SC-CM) or with peptides cleaved by CNBr from intact virions (SP-CM). CHF activity stimulated by both antigens reached a maximum after day 3 of culture. In contrast, interleukin 2 (IL 2) activity peaked at day 2 and had essentially disappeared by day 4. Fractionation of day-4 SC-CM and SP-CM preparations by gel filtration revealed peaks of activity at apparent m.w. of 17,000 (CHF17) and 30,000 (CHF30). Under certain conditions, a peak of CHF activity appeared in the void volume with an apparent m.w. of 75,000 or greater. These results indicate that CHF activity is mediated by molecules distinct from IL 2.     

The murine interleukin 2 receptor. IV. Biochemical characterization

The IL 2 receptor isolated from the IL 2-dependent CTL-L cell line was subjected to biochemical analysis. Pulse-chase and tunicamycin studies, as well as digestion with the endoglycosidases, Endo-F and Endo-H, of 35S-methionine-labeled IL 2 receptors suggested a single protein precursor of 32,000 (p32) daltons. The p32 precursor was rapidly processed by addition of high-mannose-containing core N-linked sugars to intracytoplasmic precursor intermediates of 38,000 (p38) and 40,000 (p40) daltons, which undergo further processing to yield a mature surface receptor with heterogeneous apparent m.w. of 52,000 to 65,000 (p58). Two-dimensional gel studies indicated that p58 exhibited broad charge heterogeneity between pH 4.6 and 6.3. Endo-F digestions of p58 shifted the isoelectric focus point to a more basic 5.5 to 7.4. This considerable charge heterogeneity is consistent with the possibility that other posttranslational modifications to the mouse IL 2 receptor occur besides addition of complex N-linked glycans. Immunoprecipitations of the IL 2 receptor from surface iodinated cells also revealed an additional band at 110,000 (p110) daltons. IEF vs SDS-PAGE two-dimensional gel studies demonstrated that p110 also had an isoelectric focus point identical to p58. Western blot studies with an anti-IL 2 receptor monoclonal antibody (7D4) demonstrated that p38, p40, p58, and p110 each expressed the epitope recognized by this antibody. Thus, it is likely that p110 is not a unique molecule that coprecipitates with the IL 2 receptor. Western blot analysis of mitogen-stimulated T and B lymphocytes also revealed bands similar to p58 and p110, although these bands had an average apparent m.w. 3000 to 6000 less than those seen for CTL-L cells.     

Purification of mouse interleukin 2 to apparent homogeneity

A procedure has been developed for the rapid purification of mouse interleukin 2 (IL2) to apparent homogeneity, using gel filtration, anion exchange, hydrophobic chromatography, and reverse phase high pressure liquid chromatography (RP-HPLC). IL2 eluted at a high concentration of acetonitrile on HPLC (approximately 40%), well removed from other proteins. This protocol did not resolve isoelectric variant forms of IL2. Both the biological activity and protein migrated as a band of apparent molecular weight 22,000-23,000 on SDS-polyacrylamide gel electrophoresis. It had a high potency, producing 30% of the maximal response in T cell growth at a concentration of 2-4 X 10(-12) M. Mouse Il2 synthesized in a wheat germ cell-free translation system behaved similarly on RP-HPLC as the form secreted by EL4 cells. Thus, the hydrophobicity of mouse IL2, which facilitates its purification, is an intrinsic property of the protein, determined primarily by its amino acid sequence.     

Multiplication-stimulating activity for chicken embryo fibroblasts from rat liver cell conditioned medium: a family of small polypeptides

A partially purified multiplication-stimulating activity for chicken embryo fibroblasts in cell culture was isolated from rat liver cell conditioned medium (see preceding paper, Dulak and Temin, 1973). It has been analyzed by isoelectric focusing and by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Multiplication-stimulating activity resided in a family of at least four polypeptides which were similar in apparent molecular size, but different in electrical charge. These polypeptides have a specific activity of about 50,000 with respect to serum. One of them has been purified on a small scale to apparent homogeneity in a sodium dodecyl sulfate polyacrylamide gel.     

Purification and properties of avian liver p-hydroxyphenylpyruvate hydroxylase.

Avian liver p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27) was purified to a 1000-fold increase in specific activity over crude supernatant, utilizing a substrate analogue, o-hydroxyphenylpyruvate, to stabilize the enzyme. The preparation was homogeneous with respect to sedimentation with a sedimentation velocity (s20,w) of 5.3 S. The molecular weight of the enzyme was determined to be 97,000 +/- 5,000 by sedimentation equilibrium, and the molecular weight of the subunits was determined to be 49,000 +/- 3,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis revealed heterogeneity of the purified enzyme. The multiple molecular forms were separable by isoelectric focusing, and their isoelectric points ranged from pH 6.8 to 6.0. The amino acid compositions and tryptic peptide maps of the three forms isolated by isoelectric focusing were very similar. The forms of the enzyme had the same relative activity toward p-hydroxyphenylpyruvate and phenylpyruvate. Conditions which are known to accelerate nonenzymic deamidation of proteins caused interconversion of the multiple molecular forms. Iron was the only transition metal found to be associated with the purified enzyme at significant levels. The amount of enzyme-bound iron present in equilibrium-dialyzed samples was equivalent to 1 atom of iron per enzyme subunit. Purification of the enzyme activity correlated with the purification of the enzyme-bound iron. An EPR scan of the purified enzyme gave a signal at g equal 4.33, which is characteristic of ferric iron in a rhombic ligand field.     

Positional Specificity and Fatty Acid Selectivity of Purified sn-Glycerol 3-Phosphate Acyltransferases from Chloroplasts

Soluble acyl-CoA:sn-glycerol 3-phosphate acyltransferases (EC 2.3.1.15) which are localized in chloroplasts were purified from leaves of Pisum sativum and Spinacia oleracea and obtained free from interfering activities. The purification raised the specific activities by factors of about 1,000 for pea and 200 for spinach preparations. In pea chloroplasts, acyltransferase activity occurs in two soluble forms with apparent isoelectric points of 6.3 and 6.6. For both forms, the same molecular weight of about 42,000 was determined. The enzyme from spinach chloroplasts showed a slightly higher molecular weight and a lower isoelectric point of 5.2.     

Purification and characterization of human leukocyte interferon components.

Human leukocyte interferon, prepurified either by acid ethanol extraction or by affinity chromatography with antibodies, was further purified by gel filtration in the presence of sodium dodecyl sulfate. Interferon was eluted from gel filtration columns as an apparently homogeneous entity with a molecular weight of 26,600, resulting in an up to 50-fold additional purification during a single step. The antiviral activity could be further resolved into two components by hydroxylapatite adsorption chromatography. The isolated components (A and B) were distinguishable by isoelectric focusing and polyacrylamide gel electrophoresis. The apparent molecular weights were 20,000 to 16,000 and 16,000, respectively. No differences were detected in their susceptibility toward reduction of disulfide bonds by beta-mercaptoethanol. Both could be obtained on a preparative scale with minimal losses in biological activity.     

Characterization of an immunosuppressive factor derived from colon cancer cells

The colon cancer cell line, HT29, produces a soluble substance (HT29 factor) that blocks mitogen-induced T cell proliferation and the production of interleukin 2 (IL 2). Inhibition of T cell proliferation by the HT29 factor is reversible and is not due to a decline in cell viability or an alteration in the kinetics of T cell proliferation. It occurs even when the HT29 factor is added only 24 hr before terminating the T cell cultures, indicating that the factor affects cell division after activation of T cells has already occurred. No inhibitory activity was found in medium conditioned by human colonic epithelial cells or fibroblasts. The factor has an apparent m.w. of 56,000 and an isoelectric point of 7.9. It is sensitive to endopeptidases, heating to 56 degrees C, and extremes of pH. The HT29 factor also suppresses IL 2 production by T cells. However, low IL 2 availability alone cannot account for the suppressive effect of the factor on T cell proliferation, because the addition of exogenous IL 2 does not reverse the inhibition. This block in IL 2 responsiveness is not primarily due to a decrease in IL 2 receptors because Tac expression on activated T cells is minimally decreased during a 24-hr exposure to the HT29 factor. In addition, IL 2-induced proliferation of mitogen-activated T cells is inhibited only slightly by the HT29 factor, indicating that a block in the interaction of IL 2 with its receptor is not its main mechanism of action. Thus the inhibition of T cell proliferation is likely to be due primarily to a mechanism independent of IL 2.     

Release of sphingomyelin phosphodiesterase (acid sphingomyelinase) by ammonium chloride from CL 1D mouse L-cells and human fibroblasts. Partial purification and characterization of the exported enzymes

In cultured human fibroblasts and mouse L-cells the lysosomotropic agent, ammonium chloride, caused release of acid sphingomyelinase into the culture medium. The water-soluble enzymes were partially purified by sequential chromatography on ConA-Sepharose, octyl-Sepharose and Sepharose CL-4B. Mouse sphingomyelinase was purified up to 64-fold and human sphingomyelinase 134-fold from the culture medium. Specific activities were 925 nmol/(h X mg) and 1 434 nmol/(h X mg), respectively. The final enzyme preparations obtained were free of other lysosomal enzyme activities tested and had very similar properties: optimal activity at pH 4.8 (mouse enzyme) and pH 4.4 (human enzyme), Km values of 6.2 X 10(-5)M and 2.4 X 10(-5)M, respectively, and an apparent molecular mass of 68 kDa. In isoelectric focusing the enzymes peaked at pH 4.78 (mouse enzyme) and pH 4.75 (human enzyme).     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

Purification of Cytochrome b5 from Pig Testis Microsomes by Isoelectric Focusing in an Immobiline pH Gradient

Testicular cytochrome b5 was purified by a procedure including preparative isoelectrofocusing. The cytochrome b5 was determined to have an isoelectric point of 4.45 on analytical isoelectric focusing. The purified cytochrome b5 was found to be homogeneous and its molecular weight was estimated to be 16,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The oxidized and reduced forms of the purified preparation exhibited absorption spectra of a typical cytochrome b5. A 69-fold purification was achieved with an overall yield of 6.2%. Following preparation of the microsomes, the purification is accomplished by a two-step procedure utilizing column chromatography and preparative isoelectric focusing.     

Characterization of recombinant human lymphotoxin (tumor necrosis factor-beta) produced by a mammalian cell line

Lymphotoxin (LT) was purified from serum-free conditioned media of a recombinant mammalian cell line transfected with human lymphotoxin cDNA. The purification scheme consisted of controlled pore glass chromatography, Q-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose chromatography. The purified protein was found to be homogeneous by reverse-phase high-performance liquid chromatography and had an approximate specific activity of 130 X 10(6) units per milligram protein as determined by the L-929 cytotoxicity assay. Purified LT had an isoelectric point of approximately 6.85 and an apparent molecular weight of 50,000 by gel permeation high-pressure liquid chromatography. However, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two distinct bands at approximate molecular sizes of 25 and 20 kDa were observed. Both the bands were immunoreactive by Western blot analysis and found to be associated with biological activity. The two forms of lymphotoxin differed from each other with respect to protein structure. Amino-terminal amino acid sequence analysis revealed that the 25-kDa LT sequence starts with Leu-Pro-Gly-residues whereas that of the 20-kDa LT begins with His-Leu-Ala; thus the latter form is truncated by 20 amino acid residues from the amino terminal. Two species of LT also differed from each other with respect to carbohydrate structure. Enzymatic removal of sialic acid reduced the molecular weight of 25 kDa by approximately 5 kDa whereas that of the 20-kDa LT was unchanged. A reduction in an apparent molecular size by approximately 4 kDa of both species of LT was observed on removal of N-linked oligosaccharides. Treatment with O-Glycanase had minimal effect on either form of LT. The recombinant lymphotoxin described here was found superior in its solubility behavior as compared to bacterial cell derived LT. Overall, mammalian cell line derived recombinant LT appears closer in its properties to natural LT than does bacterial cell derived recombinant LT.     

Soluble cAMP-independent protein kinase from human spleen

A protein kinase (EC 2.7.1.37) was purified 2000-fold, from the soluble protein fraction of human spleen cells, using ion-exchange chromatography, ammonium sulfate fractionation, and gel filtration. This rapid procedure yielded 30% of the initial activity and an enzyme preparation with specific activity of 62 nmol min-1 mg-1 of protein. On the basis of disc gel electrophoresis in sodium dodecyl sulfate acrylamide gels and isoelectric focusing the enzyme preparation appears homogeneous and to consist of one polypeptide with a molecular weight of 43,000 and having a pI of 7.1. The purified enzyme activity is cyclic AMP and cGMP independent phosphorylates both alpha-casein and phosvitin, and uses Mg2+ ATP and Mg2+ GTP as phosphate donors, exhibiting an apparent Km of 2.0 and 6.6 X 10(-5)m, respectively. Furthermore, the enzyme activity is strongly inhibited by heparin (K50 = 0.1 micrograms/ml). These catalytic properties are characteristic of the enzyme casein kinase II, as described in several eukaryotic cells.