The relationship between Mg(2+)-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, IN(CHAPS) and IN(dial), were purified in the presence of detergent, but in the case of IN(dial), the detergent was removed during a final dialysis. The third preparation, IN(zn), was purified without any detergent. The three preparations displayed comparable Mn(2+)-dependent activities. In contrast, the Mg(2+)-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. IN(CHAPS) was not capable of using Mg(2+) as a cofactor, whereas IN(zn) was highly active under the same conditions. In the accompanying paper [Deprez, E., et al. (2000) Biochemistry 39, 9275-9284], we used time-resolved fluorescence anisotropy to demonstrate that IN(CHAPS) was monomeric at the concentration of enzymatic assays. Here, we show that IN(zn) was homogeneously tetrameric under similar conditions. Moreover, IN(dial) that exhibited an intermediary Mg(2+)-dependent activity existed in a monomer-multimer equilibrium. The level of Mg(2+)- but not Mn(2+)-dependent activity of IN(dial) was altered by addition of detergent which plays a detrimental role in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase to use Mg(2+) as a cofactor is related to its self-assembly state in solution, whereas Mn(2+)-dependent activity is not. Finally, the oligomeric IN(zn) was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric IN(CHAPS) strictly required Mn(2+). Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg(2+).
We have previously reported that a neutral glycolipid (globotriosylceramide; Gb3) was specifically expressed on Burkitt's lymphoma cells and on a subset of germinal center tonsillar B lymphocytes. Recently the Gb3 molecule was recognized as a new B cell differentiation antigen and now defines the CD77 cluster. Here we report an extensive phenotypic and functional characterization of the tonsillar CD77+ B lymphocytes. These cells have a low buoyant density and are thus purified using a Percoll gradient. They express various B cell antigens such as CD19, CD20, CD21, CD22 and CD40, as well as the adhesion molecules LFA-1, LFA-3 and CD44. They are positive for surface IgM and negative for surface IgD. Although these results suggest a phenotype of activated B cells, the CD77+ cells are negative for the classical activation antigens: CD23 (the low-affinity Fc receptor for IgE), CD25 [the interleukin (IL) 2 receptor alpha chain] and CD71 (the transferrin receptor). Proliferation and protein synthesis of CD77+ cells was measured after stimulation with a range of mitogens and IL. None of the agents tested are able to induce proliferation and protein synthesis with the exception of a combination of recombinant IL 4 plus anti-CD40 antibody. When examined by electron microscopy, CD77+ B lymphocytes present a morphology similar to that of cells undergoing programmed cell death, also called apoptosis (i.e. chromatin condensation, nuclear fragmentation, membrane blebbing). As shown by direct examination of DNA, these CD77+ cells are indeed in the process of apoptosis. Treatment of the CD77+ cells by recombinant IL 4 and anti-CD40 antibody prevents apoptosis. All these results suggest that the CD77 molecule defines a B lymphocyte maturation pathway, specific for germinal center, where the cells undergo programmed cell death.
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