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The binding of [3H]tuftsin to normal and in vivo stimulated mouse peritoneal macrophage populations was studied at 22 degrees C. The [3H]tuftsin binding to thioglycollate-stimulated macrophages was shown to be rapid and saturable, with an equilibrium dissociation constant (K(D)) (calculated from a Scatchard plot) of 5.3 X 10(-8) M. The calculated number of binding sites per macrophage amounts to approximately 72,000. Binding competition studies with unlabelled tuftsin yielded a K(D) of 5.0 X 10(-8) M. [3H] [N-Acetyl-Thr1]tuftsin, an inactive analog of tuftsin, failed to bind specifically to thioglycollate-stimulated macrophages. [N-Acetyl-Thr1]tuftsin and the tripeptide [Des-Arg4]tuftsin failed to compete for tuftsin binding sites, while [D-Arg4]tuftsin, an analog with small tuftsin-like activity, exhibited a low degree of inhibition of [3H]tuftsin binding. Thus a rather high degree of specificity is involved in the binding of the tetrapeptide. Normal as well as six different macrophage populations induced by stimulation with thioglycollate, concanavalin-A, starch, mineral oil, glucan and Bacillus Calmette Guerrin (BCG), exhibited a similar degree of binding of [3H]tuftsin. Corynebacterium parvum (CP)-stimulated macrophages, on the other hand, showed a 6- to 10-fold-lower capacity for tuftsin binding. Under similar experimental conditions, mouse fibroblast and lymphocyte preparations revealed no detectable specific binding. Tuftsin augmented the phagocytic response of normal and stimulated macrophages assessed both for phagocytosis mediated via the Fc-receptor and via non-specific receptors. CP-stimulated macrophages did not exhibit an increased phagocytic response upon treatment with tuftsin.

The immunoglobulin heavy-chain-associated tetrapeptide, tuftsin (Thr-Lys-Pro-Arg), known for its phagocytosis-stimulating activity, was found to augment the antigen-specific, macrophage-dependent education of T lymphocytes.The investigation of stereospecific characteristics of the tetrapeptide, by use of structural analogs with different modifications, revealed strict structural requirements for eliciting the immunogenic activity of macrophages. It was found that the most important moiety for its activity is the dipeptide ProArg. Ths finding is of interest in view of the appearance of this particular dipeptide in other bioregulatory peptides, including many of the peptide hormones. The significance of the appearance of a commoil structure in such molecules, which may act through specific receptors on different target cells, is discussed. Numerous studies have demonstrated the central role of T cells in humoral and cellular immune responses. In addition to functioning as effector cells, T cells may also augment as helpers or suppress the function of other cellular components of the immune system (1). The activation by antigen of T cells, resulting in antibody production, seems to depend on the presence of macrophages (2-5). The presentation of antigen via the macrophage determines the triggering and generation of an active T cell (4, 6). Yet, the mechanism that controls the activation of the immunogenic potential of the macrophages themselves is largely unknown.Studies of Najjar and his coworkers (7,8) and those carried out in our laboratory (9, 10) showed that the basic tetrapeptide

The major industrial use of thermostable and detergent compatible proteinases is in laundry detergent formulations. ' Detergents such as Era Plus@ (Procter and Gamble), Tide@ (Colgate Palmolive), and Dynamo@ (Procter and sulfate precipitation followed by separation of proteinase R and T from contaminants and each other by ion exchange chromatography.Gamble) contain proteolytic enzymes, the majority of which are produced by the members of the genus Bacillus. Although subtilisins have been the enzymes of choice for detergent formulations (US patent Nos. 1240058, 374971, 370482, and 4266031, and UK patent No. 13155937), these are not the ideal detergent enzymes due to low thermal stability in the presence of detergents and short halflife on the shelf. Recently, modified subtilisins have been generated with greater thermal stability by means of site directed mutagenesis te~hnique.~-~ In this report, the laundry detergent compatibility of two novel enzymes6.' from the fungus Tritirachium album Limber is described. The amino acid sequences of these two proteinases already contain some of the changes recently engineered into subtilisins. These two proteinases, termed as proteinase R and T, were extremely stable in the presence of detergents at high temperature. We found the thermal and detergent stabilities of proteinase R to be very similar to those of proteinase K which were much higher than that of subtilisin BPN'. On the other hand, proteinase T demonstrated superior stability to that of proteinase R and K. An earlier report of this work has been published as a meeting abstract.* MATERIALS AND METHODS Determination of Proteinases ActivityN-Succinyl-alanyl-alanyl-prolyl phenyl alanyl nitroanilide' was used as the substrate to monitor the production of proteinases during liquid culture while azoalbumin and azocasein were used as substrates in stability studies." To a final volume of 500 pL, 20 p L of azocasein or azoalbumin (a 5% solution in 0.W Tris HCl, pH 7.5, 1 mM CaCl,), 20 pL of enzyme (1-10 pg) and 460 pL of 50 mM Tris HC1, pH 7.5, were added. Following the incubation for 30 min at 37"C, 500 p L of 10% TCA was added and the samples were kept on ice for 15 min. After centrifugation 800 p L of supernatant was added to a tube containing 200 pL of 1.8N NaOH. The optical density was measured at 420 nm against the proper control. Stability StudiesProteinases at a concentration of 0.2 mg/mL were incubated at 50°C in the presence of different detergents. After specific intervals 10 p L of the sample were removed to determine the residual activity at 37°C using azocasein as the substrate in excess (see above). RESULTS AND DISCUSSION Purification of ProteinasesThe physical properties of the proteinases have been described e l~e w h e r e .~.~ Briefly, the molecular weights of proteinase R and T were 30,000 and 33,000 daltons, respectively. The isoelectric point of proteinase R was 9.8 while that of proteinase T was 4.5. The pH optimum of the proteinase R was between 7.0 and 10.0 while proteinase T was maximally active b...