Early pregnancy factor (EPF), first discovered in this laboratory, was described originally as a substance appearing in maternal serum within 24 h of fertilisation in all mammalian species tested. Studies have revealed that EPF is not only indicative of but also necessary for embryonic well-being. Recent studies indicate that EPF is not confined to pregnancy but is also important as a growth regulator of transformed, neoplastic and normal cells where it is required for cell proliferation; with tumour cells and trophoblast cells, it appears to function as an autocrine factor. This is in contrast to the role of an immunosuppressant assigned by virtue of its ability to stimulate from lymphocytes the release of lymphokines, shown in mice to suppress the delayed-type hypersensitivity (DTH) reaction. Our research group purified this protein from several sources and determined seventy percent of the EPF amino acid sequence. This was found to be identical to the sequence for rat chaperonin1O (cpn 10), the first fully characterised mammalian cpn1O, which belongs to the 10 kDa heat shock protein (hsp) family. Chaperonin1O is a mitochondrial matrix protein that functions as a chaperone along with chaperonin 60 (cpn60). The present study was designed to give insight into the nature of the putative receptor(s) or associate(s) of EPF on the surface of lymphocytes by answering the question: Is cpn60 the extracellular target molecule for EPF? We have developed a new double antibody sandwich ELISA for EPF/cpn1O that allows a reasonably accurate estimate of the concentration of iodinated EPF/cpn1O. Using this immunoassay, we have determined the Iodo-gen technique as the best for direct iodination of the molecule. This method places the label at the C-terminal end of the molecule and does not appear to alter its biological activity or its antibody-binding characteristics. Labelling and cross-linking experiments with neoplastic lymphocytes, MOLT-4cells, have established the presence of EPF on the surface of these cells, revealing the existence of a specific cross-linked complex of approximately 43 kDa. This complex was observed using several cross-linkers (DSS, ASA, SASD and BASED). The crosslinkers SASD and BASED also revealed larger complexes, from 60 to >94 kDa, after cross-linker cleavage. Treatment with DTT resulted in an increase in the intensity and number of bands suggesting the existence of a disulphide linked complex that is excluded from the gel. The smaller complexes observed with DSS and ASA might be part of a larger structure. EPF was also found to be associated with the surface of normal lymphocytes and a truncated form of the molecule was evident in a specific manner, being absent from CD4+-depleted human lymphocyte and monocyte populations. Under conditions capable of resolving large molecular weight molecules, a large cross-linked complex was observed (>200 kDa) using a long cross-linker such as BASED. Smaller complexes, particularly one of similar size to the 43 kDa seen with MOLT-4 cells, were observed when using short cross-linkers such as DSS. Formation of complexes was inhibited with depletion of CD4+ T cells from the lymphocyte population, fixation of lymphocytes or addition of excess unlabelled EPF/cpn1O during labelling. Parallel studies were carried out on supernatants recovered after incubation of lymphocytes with iodinated EPF/cpn1O and immunoprecipitated with two different anti-EPF/cpn1O peptide antibodies, an anti-N terminal peptide antibody and an antibody against an internal peptide (anti-I), and with a control antibody. Results indicated that the truncated form is evident only with the anti-I peptide antibody. This suggested that the cleavage of a fragment at the N-terminus of the molecule by an ectoprotease. Further experiments are required to confirm this and to determine whether the cleavage event in itself is the critical event or whether cleavage creates further active species. All these results suggest that EPF is predominantly associated with a molecule which forms a large disulphide-linked oligomer on the cell surface. The monomeric size of this molecule is still unclear although experiments with MOLT-4 cells suggest it could be approximately 100 kDa. In addition, there may be other associates of smaller size (30-50 kDa) situated in close proximity to bound EPF. Our results are consistent with a cell-surface mechanism associated with a specific cell population. In an experiment designed to address, directly, the original question of whether cpn60 is the extracellular target of EPF, anti-cpn60 antibodies did not recognised the cross-linked complex formed between EPF/cpn1O and the cell surface moiety on lymphocytes, even though anti-cpn60 antibodies were able to detect a cpn60- EPF/cpn10 complex under identical experimental conditions. Anti-cpn60 antibodies did not exhibit activity in the EPF bioassay and did not affect the activity of EPF/cpn1O samples. Therefore it seems that cpn60 is not involved in the complex formed between EPF/cpn1O and the lymphocyte surface, although the presence of cpn60 on the surface of cells, in at least some circumstances, has been described. From the present study it can be concluded that cpn60 does not appears to be the target of extracellular EPF on the surface of lymphocytes. A very interesting partial picture of putative associates of EPF has emerged, along with the recognition that association of EPF with these cells results in cleavage of the molecule. CD4+ T cells are necessary for binding and processing of EPF but it is not yet clear whether they alone are sufficient. The definitive identification of EPF associates on lymphocytes and the precise mechanism of action of EPF on these cells will be the subject of future studies.