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A 45-kDa human T cell surface glycoprotein which is tightly bound in the membrane of the resting T cell is released into the cell medium in soluble form after cell growth activation by phytohemagglutinin or neuraminidaseigalactose oxidase treatments. In limited proteolysis by Staphylococcus aureus V8 protease, two major 35-kDa and 27-kQa peptide fragments of the surface-iodinated 45-kDa protein are common to the membrane-bound and the released forms, but a third 18-kDa fragment is observed exclusively with the released protein. The apparent molecular masses of the deglycosylated peptide backbones of the membrane-bound and the released molecule are 30 jI 1 kDa, although a small size difference cannot be excluded. A polyclonal rabbit anti-(T cell membrane protein) antiserum precipitates the 45-kDa protein. A monoclonal anti-(45-kDa protein) antibody precipitates the membrane-bound 45-kDa protein solubilized with octyl glucoside, but does not precipitate the released protein. In cell culture assays, the monoclonal anti-(45-kDa protein) antibody specifically enhances the cell proliferative responses in phytohemagglutinin-treated and mixed lymphocyte cultures. These observations suggest that the 45-kDa protein has a specific receptor function in the regulation of cell proliferative responses.T cells can be activated by specific dhemical signals received at the cell surface: for example, the monoclonal anti-(T3 protein) antibody has a polyclonal mitogenic effect, whereas other monoclonal antibodies may have no or different effects on cell proliferation [I]. A specific effect of a given monoclonal antibody demonstrates a functional role of the membrane antigen recognized, even if the biochemical mechanism remains unknown ; cross-linking, modulation, uptake or shedding of the antigen, activation of enzymes or ion channels, all may play a role.In T cells, well-defined inductions of polyclonal cell proliferation by the treatment with mitogenic lectins (for a review see [ 2 ] ) or enzymes (e.g. [3]) are known which are models for the antigen-specific activation and clonal expansion. Recently, at least some of the molecules involved in the antigen recognition by cytotoxic T cells were characterized. Scveral clonotypic monoclonal antibodies appear to react with that part of the T cell receptor which determines the antigen specificity [4 -61. They precipitate a disulfide-linked heterodimeric protein of 80 -90 kDa. The 20-kDa T3 glycoprotein is co-modulated with the heterodimer, and therefore, some association between these molecules must exist [5]. It has been proposed that temporary or even long-lived protein complexes may exist at the cell surface into which the T3 and clonotypic molecules are integrated and which control cell proliferation [ 5 ] .It is to be expected that additional surface proteins regulate the cell responses to differentiation and growth-promoting signals. We have previously characterized a 45-kDa glyco-

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The sequence of the gyrase B subunit gene from Staphylococcus aureus strains resistant to the gyrase B subunit inhibitors cyclothialidine, coumermycin, and novobiocin has been determined. The residues altered in the resistant gyrase B subunits map to the ATP-binding region, suggesting that the drugs inhibit ATP binding and hydrolysis. The pattern of cross-resistances indicates that the detailed binding mode of the compounds differs.

We investigated how cyclothialidine (Ro 09-1437), a novel DNA gyrase inhibitor belonging to a new chemical class of compounds, acts to inhibit Escherichia coli DNA gyrase. Cyclothialidine up to 100 ,ug/ml showed no effect on DNA gyrase when linear DNA was used as a substrate. Under the same conditions, quinolones, which inhibit the resealing reaction of DNA gyrase, caused a decrease in the amount of linear DNA used. No effect of cyclothialidine was observed on the accumulation of the covalent complex of DNA and the A subunit of DNA gyrase induced by ofloxacin in the absence of ATP. The effect of cyclothialidine on the DNA supercoiling reaction was antagonized by ATP, reducing the inhibitory activity 11-fold as the ATP concentration was increased from 0.5 to 5 mM. Cyclothialidine competitively inhibited the ATPase activity of DNA gyrase (Ki = 6 nM). The binding of [14C]benzoyl-cyclothialidine to E. coli gyrase was inhibited by ATP and novobiocin, but not by ofloxacin. These results suggest that cyclothialidine acts by interfering with the ATPase activity of the B subunit of DNA gyrase. Cyclothialidine was active against a DNA gyrase resistant to novobiocin, suggesting that its precise site of action might be different from that of novobiocin.DNA gyrase is a type II DNA topoisomerase that catalyzes the negative supercoiling of DNA in prokaryotes. Its function is essential to DNA replication, transcription, and bacteriophage X integrative recombination (for reviews, see references 6, 14, and 27). A large body of evidence indicates that topoisomerases, including DNA gyrase, are the targets of therapeutically useful antibacterial and antitumor agents (5). The enzyme from Escherichia coli consists of two subunits, A and B, with molecular masses of 97,000 and 90,000 Da, respectively; the active enzyme is an A2B2 tetramer complex. Mechanistic studies have suggested that the following steps are involved in the DNA supercoiling process. The enzyme binds to DNA and forms a complex in which about 120 bp of DNA is wrapped around a protein core. Both strands of the wrapped DNA are then cleaved and covalent bonds are formed between the protein and the DNA (cleavable complex). A segment of DNA is passed through this break, and probably through part of the protein itself. Although gyrase requires the hydrolysis of ATP for the supercoiling of DNA, in its absence gyrase can relax negatively supercoiled DNA, albeit inefficiently. Replacement of ATP by the nonhydrolyzable ATP analog 5'-adenylyl-P--yimidodiphosphate (ADPNP) results in limited supercoiling by gyrase, suggesting that ATP binding can promote a single round of supercoiling, but the hydrolysis step is required to regenerate the enzyme in an active form (25).DNA gyrase is the target of two classes of antibiotics: the synthetic quinolones, typified by nalidixic acid and the new fluoroquinolones, and the natural coumarins, such as novobiocin and coumermycin Al. Recently, three antibacterial agents, cinodine, microcin, and clerocidin, which fall outside the quinolone and coum...