sodium-dodecyl-sulfate and Fibrosarcoma

Two kinds of arginine deiminase (AD, EC 3.5.3.6) were purified from cell extracts of Mycoplasma arginini (a-AD) and Mycoplasma hominis (h-AD), and their enzymic properties and anti-tumor activities were compared. The a-AD enzyme strongly inhibited the growth of mouse hepatoma cell line MH134 in vitro, and its concentration required for 50% growth inhibition (IC50) was estimated to be about 10 ng/ml. The IC50 value of h-AD against the same cell line was estimated to be about 100 ng/ml, due to its low enzyme activity under the physiological pH condition, i.e., pH 7.4. These results show that the reaction pH profile of the a-AD was superior to that of the h-AD as an anti-tumor enzyme. Moreover, the effects of L-arginine metabolism-related substances on the anti-tumor activity of the a-AD were examined to study the growth-inhibitory mechanism of this enzyme. The addition of 2 or 4 mM L-arginine restored, in a dose-dependent manner, the growth of mouse MH134 hepatoma and Meth A fibrosarcoma cell lines that had been inhibited by 20 ng/ml of the a-AD. The addition of 2 or 4 mM L-ornithine, which is biosynthesized from L-arginine in the urea cycle and is the starting material in the polyamine-biosynthesis pathway, also partially restored it in a dose-dependent manner. These results indicate that the tumor cell growth inhibition caused by a-AD originates from the depletion of the essential nutrient L-arginine, and that the resulting block of the polyamine-biosynthesis pathway is involved in part in the inhibitory mechanism.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Arginine; Cell Division; Electrophoresis, Polyacrylamide Gel; Fibrosarcoma; Hydrolases; Liver Neoplasms, Experimental; Mice; Molecular Sequence Data; Molecular Weight; Mycoplasma; Sequence Homology, Amino Acid; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.

Topics: Amino Acid Sequence; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cold Temperature; Drug Stability; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Fibrosarcoma; Humans; Kinetics; Molecular Sequence Data; Molecular Weight; Plasminogen Activators; Plasminogen Inactivators; Recombinant Proteins; Sodium Dodecyl Sulfate; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Human Hep G2 hepatoma and HT 1080 fibrosarcoma cells were cultured in large scale under conditions which allowed enhanced secretion of plasminogen activator inhibitor-1 (PAI-1). A modified urokinase was obtained by reacting urokinase with phenylmethylsulfonyl fluoride followed by alkali treatment. The resulting product, called anhydrourokinase, was found to reversibly bind the PAI-1 when immobilized on cyanogen bromide-activated Sepharose 4B beads. Using this affinity absorbent, we have purified PAI-1 from the cell-conditioned media. A number of differences have been observed during Hep G2 and HT 1080 PAI purification. 1) The PAI activity in Hep G2 medium concentrate is more stable, and the concentrate depleted of active PAI-1 showed spontaneous regeneration of PAI-1 activity. In contrast, the PAI activity in HT 1080 medium concentrate declines rapidly on standing. 2) Hep G2 PAI-1 invariably copurified with an adhesive protein, vitronectin or its NH2-terminal fragment, while pure HT 1080 PAI-1 alone was obtained by affinity purification on anhydrourokinase-Sepharose 4B. 3) Based on specific activity measurement and complex formation analysis using a sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis technique, the purified Hep G2 PAI-1 appears completely active while the HT 1080 PAI-1 is only one-fourth as active. SDS was found to exert dual effects on purified PAI-1s. SDS treatment partially inactivated a fully active Hep G2 PAI-1 and a moderately active HT 1080 PAI-1 but partially activated an HT 1080 PAI-1 whose activity had previously been allowed to decay to a very low level. Purified vitronectin was found to enhance and stabilize the PAI-1 activity of the partially active HT 1080 PAI-1. It is concluded that fully active PAI-1 in association with vitronectin can be isolated by anhydrourokinase-Sepharose 4B chromatography and that vitronectin is a binding protein for PAI-1 which activates and stabilizes PAI-1.

Topics: Carcinoma, Hepatocellular; Chromatography, Affinity; Culture Media; Drug Stability; Electrophoresis, Polyacrylamide Gel; Enzymes, Immobilized; Fibrosarcoma; Glycoproteins; Humans; Liver Neoplasms; Molecular Weight; Phenylmethylsulfonyl Fluoride; Plasminogen Activators; Plasminogen Inactivators; Sodium Dodecyl Sulfate; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Vitronectin

The tumor antigen capable of inducing tumor resistance (tumor rejection antigen; TRA) was separated and some of its physicochemical properties were characterized. Cytosol and plasma membrane fractions were separated from Rous sarcoma virus (RSV)-induced CSA1M tumor cells. Immunization with membrane but not cytosol fraction of these tumor cells together with complete Freund's adjuvant resulted in complete protection against subsequent challenge with viable CSA1M cells. The TRA activity contained in the membrane fraction was recovered in the sodium dodecyl sulfate (SDS)-solubilized fraction after the SDS-extraction of CSA1M membranes. This CSA1M SDS-solubilized preparation gave protection against syngeneic RSV-induced CSA9F tumor cells as well as the homologous tumor cell type, but failed to induce resistance to RSV-unrelated tumor cells. The membrane or SDS-solubilized fraction from RSV-unrelated tumor cells was unable to generate anti-CSA1M protective immunity. Physicochemical analyses have demonstrated that TRA activity in the SDS-solubilized fraction was completely abolished by treatment with proteinase K but was only marginally affected after treatment with glycosidase mixture. When the SDS-solubilized preparation was applied to a Sephacryl S-300 superfine column, TRA activity was recovered in the range of molecular weight of 50-90 kD. Further fractionation of this TRA-positive fraction by SDS-polyacrylamide gel electrophoresis revealed that the molecular size of TRA is 56-68 kD. These results indicate that membrane proteins which were isolated from CSA1M tumor cells and have a molecular size of about 60 kD are capable of inducing RSV-induced tumor-specific in vivo protective immunity.

Topics: Animals; Antigens, Neoplasm; Antigens, Surface; Avian Sarcoma Viruses; Cell Fractionation; Cell Line; Cell Transformation, Viral; Endopeptidase K; Epitopes; Fibrosarcoma; Graft Rejection; Growth Inhibitors; Male; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Sarcoma, Experimental; Serine Endopeptidases; Sodium Dodecyl Sulfate

Plasminogen activator inhibitor (PAI) purified from human placenta was compared to PAI purified from conditioned cell culture fluid of the human fibrosarcoma cell line HT-1080. The two inhibitors had a similar mobility (Mr approximately 50,000) in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Purified placental inhibitor revealed 2 major and 1 minor Coomassie blue stainable bands, while the fibrosarcoma inhibitor appeared as one band. By immunoblotting analysis both monoclonal and polyclonal antibodies against each of the inhibitors showed reaction with the inhibitor against which they were raised, but not cross reaction with the other inhibitor. Similar results were obtained, when antibody binding was tested by ELISA with the inhibitors coated on the solid phase. HPLC fingerprint patterns of cyanogen bromide fragments of the two inhibitors were different. The inhibitory activity of the placental PAI was decreased by a factor of 3 after incubation with SDS, while that of the fibrosarcoma PAI was increased by a factor of 30. It is concluded that the two inhibitors show no detectable common antigenic determinants and most likely are products of different genes.

Topics: Antibodies; Antibodies, Monoclonal; Binding Sites, Antibody; Cell Line; Chromatography, High Pressure Liquid; Collodion; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Fibrosarcoma; Glycoproteins; Humans; Paper; Placenta; Plasminogen Inactivators; Sodium Dodecyl Sulfate

Topics: Animals; Carcinoma, Ehrlich Tumor; Dose-Response Relationship, Drug; Female; Fibrosarcoma; Lysophosphatidylcholines; Male; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Sarcoma, Experimental; Sodium Dodecyl Sulfate; Surface-Active Agents; Time Factors