alpha-chymotrypsin and Carcinoma--Ehrlich-Tumor

Topics: Animals; Arginase; Asparaginase; Carcinoma 256, Walker; Carcinoma, Brown-Pearce; Carcinoma, Ehrlich Tumor; Carcinoma, Krebs 2; Chymotrypsin; Deoxyribonucleases; Enzyme Therapy; Glutaminase; Hyaluronoglucosaminidase; Leukemia, Experimental; Leukemia, Radiation-Induced; Lyases; Lymphoma, Non-Hodgkin; Mammary Neoplasms, Experimental; Mice; Neoplasms, Experimental; Pyridoxal Phosphate; Rats; Ribonucleases; Salts; Sarcoma, Avian; Sarcoma, Experimental; Trypsin; Vanadium

Topics: Amylases; Animals; Arginase; Benz(a)Anthracenes; Carcinoma; Carcinoma 256, Walker; Carcinoma, Brown-Pearce; Carcinoma, Ehrlich Tumor; Catalase; Chymotrypsin; Deoxyribonucleases; Enzyme Therapy; Humans; Hyaluronoglucosaminidase; Mammary Neoplasms, Experimental; Mice; Neoplasms, Experimental; Osteosarcoma; Peroxidases; Rats; Ribonucleases; Sarcoma; Sarcoma 180; Sarcoma, Experimental; Trypsin

Polymeric doxorubicin prodrugs were prepared linking monomethoxy poly(ethylene glycol), 5000 D molecular weight, to the doxorubicin amino group, using an amino acid or a peptide as a spacer arm. As spacers glycine, L-phenylalanine, L-tryptophan and glycil-L-valil-L-phenylalanine were used. The conjugates showed enhanced stability to alkaline degradation compared to the free doxorubicin. Towards Ehrlich solid tumor in mice the glycin spaced derivative was devoid of activity, whereas the phenylalanine and tryptophan derivatives were 20% and 16% active and the tripeptide one 50% active with respect to free doxorubicin. On the other hand the derivatization was accompanied by a great decrease of toxicity in mice with respect to the free drug. Doxorubicin was not released from conjugates by chymotrypsin incubation or in plasma.

Topics: Amino Acid Sequence; Amino Acids; Animals; Carcinoma, Ehrlich Tumor; Chymotrypsin; Doxorubicin; Hydrolysis; Magnetic Resonance Spectroscopy; Mice; Mice, Inbred Strains; Molecular Sequence Data; Peptides; Polyethylene Glycols; Spectrophotometry, Ultraviolet

The ornithine decarboxylase-inducing factor (ODC factor) was purified about 1,000-fold in 42% yield from the ascites fluids of an Ehrlich ascites tumor by a combination of centrifugation and concanavalin A (ConA) treatment. A single ip injection of 0.5 micrograms of the purified factor per mouse resulted in half-maximum induction of liver ODC. The factor was found to be a trypsin- and chymotrypsin-resistant, acidic glycoprotein (pI about 4.43) with a minimum molecular weight of about 70 kilodaltons, containing a disulfide bond(s) in its functional domain. It did not react with ConA. This factor induced retrodifferentiation of liver function, causing a marked increase of prototype M2 isozyme of pyruvate kinase. It reduced liver catalase activity, and also modified thyroid hormone metabolism, reducing the serum levels of T4 and T3. These results suggest that the ODC factor is multifunctional and induces many of the changes observed in a tumor-bearing host.

Topics: Animals; Carcinoma, Ehrlich Tumor; Chromatography, Affinity; Chymotrypsin; Enzyme Induction; Glycoproteins; Isoenzymes; Kinetics; Liver; Male; Mice; Mice, Inbred ICR; Ornithine Decarboxylase; Poly A; Pyruvate Kinase; RNA; RNA, Messenger; Thyroxine; Triiodothyronine; Trypsin

Ribonucleotide reductase catalyzes the rate-limiting step in the formation of 2'-deoxyribonucleoside 5'-triphosphates. It consists of two nonidentical protein subunits, the nonheme iron subunit, and the effector-binding subunit. It has previously been shown that these two components making up the active enzyme species are not coordinately synthesized or degraded. It was found that the effector-binding subunit was more sensitive to proteolysis by chymotrypsin, to heating at 55 degrees C, and to the sulfhydryl reagents, pCMB and NEM. The nonheme iron subunit was more sensitive to trypsin treatment. ATP and dATP protected the effector-binding subunit from proteolytic inactivation. Neither ATP nor CDP protected the effector-binding subunit from inactivation by the sulfhydryl reagents. These data indicate that the protein properties of the two subunits of mammalian ribonucleotide reductase are significantly different.

Topics: Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Chymotrypsin; Hot Temperature; Mice; Mice, Inbred ICR; Peptide Hydrolases; Ribonucleotide Reductases; Sulfhydryl Reagents; Trypsin

The spatial arrangement of the subunits of RNA polymerase II from Ehrlich ascites tumor cells was investigated by measuring the sensitivity of each subunit in the native enzyme to various proteinases. The results showed that the largest two subunits (a and b) were sensitive to all the proteinases tested, whereas two smaller subunits (e and h) were resistant to these enzymes. These results suggest that in the native enzyme subunits e and h are located in the inside of RNA polymerase II, forming a core. It was also found that the conformation of the DNA binding subunit a changes when the enzyme binds to DNA, and it becomes much more susceptible to chymotryptic digestion.

Topics: Animals; Binding Sites; Carcinoma, Ehrlich Tumor; Chymotrypsin; DNA; Electrophoresis, Polyacrylamide Gel; Macromolecular Substances; Peptide Hydrolases; Protein Conformation; RNA Polymerase II

The structural relationships of S-II, S-II', and S-I(b) stimulatory proteins of RNA polymerase II purified from Ehrlich ascites tumor cells were investigated. From analysis of the amino acid compositions and tryptic peptide maps of these proteins labeled with radioiodinated Bolton-Hunter reagent, it was concluded that S-I(b) is a part of S-II located at either the amino- or carboxyl-terminal and that only this region mainly contains radioiodinatable amino acid residues when labeled using 125I. On chymotryptic digestion, S-II was cleaved to 21- and 18-kDa fragments in the presence of DNA. The 21-kDa fragment was found to be sufficient for stimulation of RNA polymerase II. It was suggested that S-II' is formed by phosphorylation of S-II in the domain containing the 18-kDa fragment.

Topics: Amino Acids; Animals; Carcinoma, Ehrlich Tumor; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Indicators and Reagents; Mice; Peptide Fragments; Protein Conformation; RNA Polymerase II; Succinimides; Trypsin

By use of purified RNA polymerase II, it was demonstrated that S-II, a stimulatory protein of RNA polymerase II, enhanced the frequency of initiation of transcription from discrete sites on the promoter region of the silk fibroin gene integrated in a supercoiled plasmid DNA in the presence of manganese. In the absence of S-II, RNA polymerase II preferentially initiated RNA synthesis from site +25, 25 bases downstream from the cap site. Of these initiation sites, the initiation from site +25 was not affected by S-II, suggesting that site +25 is structurally different from other initiation sites, including the cap site, and that S-II modifies the latter sites to the same structure as that of site +25. Direct interaction between S-II and DNA in the initiation complex was shown by demonstrating a conformational change of S-II on its interaction with DNA; namely, S-II in the initiation complex was as sensitive to chymotryptic digestion as S-II interacting with DNA, whereas free S-II was completely insensitive to chymotryptic digestion.

Topics: Animals; Carcinoma, Ehrlich Tumor; Chymotrypsin; DNA, Neoplasm; Fibroins; Promoter Regions, Genetic; RNA; RNA Polymerase II; Transcription, Genetic

Media conditioned by tumor cells were studied for the presence of factor(s) that increase the rate of random migration (chemokinesis) of Corynebacterium parvum-activated macrophages. A capillary tube assay was developed and utilized to expediently monitor the chemokinetic activity of macrophages incubated in whole and fractionated media. Media conditioned by six different syngeneic and allogeneic mouse tumor cell lines demonstrated significantly higher chemokinetic activity than unconditioned or normal fibroblast conditioned media. The chemokinetically active component of the Lewis Lung conditioned media was found to be a trypsin sensitive, heat stable, high molecular weight (300,000-480,000 dalton range) factor that had no chemotactic (directional migration) activity. Pyran-activated macrophages also responded chemokinetically to the Lewis Lung factor while oyster glycogen and thioglycolate-elicited macrophages did not. The similarity and differences between the physical properties of the chemokinetic factor, other migration stimulating factors, and tumor-associated proteins are discussed.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Line; Cell Movement; Chemotactic Factors; Chemotaxis; Chymotrypsin; Fibrosarcoma; Lung Neoplasms; Macrophages; Male; Mammary Neoplasms, Experimental; Melanoma; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Trypsin

Biphasic kinetic data were obtained when trypsin (EC 3.4.21.4) which had previously been complexed with a thiol-containing inhibitor (present in Ehrlich ascites tumour cells) was incubated with incremental additions of periodate. At low concentrations of periodate the trypsin was re-activated whilst at higher concentrations of periodate the trypsin was irreversibly inhibited. This biphasic reactivation followed by inhibition was also demonstrated when trypsin was first inhibited by dithiothreitol and followed by incremental addition of periodate. Similar results were obtained with chymotrypsin (EC 3.4.21.1). Incremental additions of either dithiothreitol or periodate caused inhibition of both these enzymes. The biphasic kinetic data can be explained in terms of reduction and oxidation of a significant disulphide bond in both trypsin and chymotrypsin which can be cleaved by thiols in a disulphide exchange reaction [1]. This bond is thought to maintain the active centres of each of these enzymes in a conformation sterically favourable for enzymic cleavage of specific peptide bonds in the protein substrates (polymeric collagen fibrils and casein) employed in this study.

Topics: Animals; Carcinoma, Ehrlich Tumor; Chymotrypsin; Dithiothreitol; Enzyme Reactivators; In Vitro Techniques; Kinetics; Mice; Periodic Acid; Trypsin Inhibitors

Topics: Animals; Carcinoma, Ehrlich Tumor; Chymotrypsin; Enzyme Activation; Kinetics; Mice; Trypsin; Trypsin Inhibitors