Affinity chromatography of trypsin and related enzymes. V. Basic studies of quantitative affinity chromatography.

A detailed study of the quantitative affinity chromatography of trypsin [EC 3.4.21.4] is reported here. Frontal chromatography using an enzyme solution of very low concentration on an affinity adsorbent gave the dissociation constant of the enzyme-immobilized ligand complex (Kd). Kd values determined under various conditions enabled us to discuss in detail the interaction of trypsin and affinity adsorbents (mainly Gly-Gly-Arg Sepharose). The pH dependence of Kd was consistent with that of the interaction of trypsin and product-type compounds. The effects of changes in temperature, ionic strength, dielectric constant, etc., were also studied. The Ki values of soluble competitive inhibitors can be determined by analysis of their effects on the elution volume of the enzyme. The values obtained were in good agreement with those obtained by kinetic analysis. The present method proved to be useful as a general procedure to investigate the interaction of a protein and a specific ligand.     

A hypotetraploid human T lymphoid cell line established by cell fusion.

A human T lymphoid cell line was established by cell hybridization technique from peripheral blood leucocytes of a patient with Sezary syndrome. The cells beared the surface antigens of human T lymphocyte specificity as demonstrated by immune cytolysis tests, but did not form E rosettes with sheep red blood cells. Isozyme patterns of enzymes in this line such as lactate dehydrogenase, glucose 6-phosphate dehydrogenase and esterase were of human type. The line had 79 chromosomes in modal number. This case supports the proposal that the production of tetraploids is favourable for establishment of cell lines.     

Random replication and random assortment model for plasmid incompatibility in bacteria

To understand the incompatibility between two related plasmids, both of which replicate in an autonomous state under a common control mechanism, we have developed a model that assumes a random choice mechanism for replication of plasmid copies and their random assortment into daughter cells upon cell division. Segregation kinetics by this model is analyzed mathematically and the number of generations required for segregation is calculated as a function of plasmid copy number per cell. The results obtained offer enough quantitative data to make our model reasonably realistic.     

Inhibition of topoisomerase I by heparin

DNA topoisomerase I isolated from mouse mammary cacinoma cells was shown to be inhibited by heparin, the dose giving 50% inhibition (IC₅₀) being 0.20 μg/ml. Other chemically related acid mucopolysaccharides including heparan sulfate, dermatan sulfate etc. were more than 500 times less active than heparin. When the amount of enzyme was doubled relative to the substrate the inhibition was reversed. Addition of heparin to assay mixtures after the initiation of the reaction immediately inhibited the enzyme reaction.     

A chaperonin from a thermophilic bacterium, Thermus thermophilus, that controls refoldings of several thermophilic enzymes.

A chaperonin has been purified from a thermophilic bacterium, Thermus thermophilus. It consists of two kinds of proteins with approximate Mr 58,000 and 10,000 and shows a 7-fold rotational symmetry from the top view and a "football"-like shape from the side view under the electron microscopic view. Its weak ATPase activity is inhibited by sulfite and activated by bicarbonate. ATP causes change of its mobility in nondenaturating polyacrylamide gel electrophoresis. The T. thermophilus chaperonin can promote in vitro refolding of several guanidine HCl-denatured enzymes from thermophilic bacteria. At high temperatures above 60 degrees C, where the native enzymes are stable but their spontaneous refoldings upon dilution of guanidine HCl fail, the chaperonin induces productive refolding in an ATP-dependent manner. No or very poor refolding is induced when the chaperonin is added to the solution aged after dilution. An excess amount of the chaperonin is inhibitory for refolding. At middle temperatures (30-50 degrees C), where spontaneous refoldings of the enzymes occur, the chaperonin arrests refolding in the absence of ATP and refolding is induced when ATP is supplemented. At temperatures below 20 degrees C, where spontaneous refoldings also occur, the chaperonin arrests the refolding but ATP does not induce refolding.     

Mechanism of induction of cellular DNA synthesis by the adenovirus E1A 12S cDNA product

The mechanism of induction of DNA synthesis in quiescent rat 3Y1 cells by the adenovirus E1A gene was investigated using the 3Y1 derivative cell lines g12-21, gn12RB1, and gn12RB2. The g12-21 cells express the E1A 12S cDNA and the latter two cells express both the E1A 12S cDNA and the human retinoblastoma susceptibility (Rb) gene at different levels in response to dexamethasone (dex). The cDNA sequences of E1A-inducible cell cycle-dependent genes, clone 3 and clone 16, were isolated by differential screening of a cDNA library constructed from dex-treated g12-21 cells. The quiescent 3Y1 cells induced c-fos and c-myc expression within 2 h after serum stimulation and expressed clone 16 and clone 3 transiently at around 8 h before the onset of DNA synthesis (10 h). In contrast, the quiescent g12-21 cells treated with dex expressed a high level of E1A at 6 to 8 h after treatment and expressed clone 16 and clone 3 at around 8 h without stimulation of c-fos and c-myc expression, suggesting that E1A bypasses the cell cycle early in G1. The half-maximal rate of DNA synthesis was reached in a much shorter time in dex-treated g12-21 cells (12 h) than in serum-treated 3Y1 cells (18 h), suggesting that E1A also bypasses the cell cycle at the G1/S boundary. The gn12RB1 and gn12RB2 cells were unable to induce DNA synthesis in response to dex presumably due to lower levels of E1A expression, although gn12RB2 but not gn12RB1 cells could express clone 16 and clone 3. These results suggest that the level of E1A required for bypass at the G1/S boundary is higher than that required early in G1.     

Amino acid sequence and disulfide-bridge location of canine haptoglobin.

The complete amino acid sequence and disulfide-bridge location of canine haptoglobin (Hp) were determined by analyzing various fragments produced chemically and/or enzymatically. Canine Hp consists of two light (L) and two heavy (H) chains with 83 and 245 amino acid residues, respectively. It has three potential oligosaccharide-binding sequences, Asn-X-Ser/Thr, one in the L chain and two in the H chain. All of them are glycosylated. Comparison of the amino acid sequences between canine Hp and human Hp shows 68 and 85% homology for L chains and H chains, respectively. About 20% of the canine L chain still retains a carboxyl-terminal arginine residue, which is completely removed during maturation in human L-chain. The half-cystine residue at position 15 in the L chain, which participates in the inter-L chain disulfide bridging in human Hp, has been replaced by a leucine residue in canine Hp. Therefore, an LH unit in canine Hp may be joined to another LH unit by a noncovalent (mainly hydrophobic) interaction to form the complete molecule. The disulfide bridges in canine Hp link Cys-34L to Cys-68L, Cys-72L to Cys-105H, Cys-148H to Cys-179H, and Cys-190H to Cys-220H, as in the case of human Hp.