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Anoikis is a specific type of apoptosis caused by the detachment of anchorage-dependent cells from their supportive matrix. Aggregation of suspended cells is believed to suppress anoikis. Here we describe the effects of cellular aggregation on anoikis in Madin-Darby canine kidney (MDCK) cells. Suspension cultures of MDCK cells grown under conditions known to induce extensive cellular aggregation were less able to reattach to culture dishes, exhibited higher caspase-8 activity, and contained more sub-G 1 cells than suspension cultures did with less cellular aggregation. When suspension cultures of MDCK cells were separated into aggregated cells and single cells, the aggregated cells had low caspase-8 activity regardless of suspension conditions, whereas the single cells had higher caspase-8 activity that increased with an increasing degree of aggregation. These results suggest that cell-cell interactions in cellular aggregates of suspended MDCK cells facilitate anoikis, causing more apoptosis in individual cells than when these interactions are absent.

Our previous work showed that the active site heterogeneity in heavy meromyosin (HMM) becomes evident when highly reactive SH-groups in HMM are modified by thimerosal (Kawamura, Higuchi, Emoto, & Tawada (1985) J. Biochem. 97, 1583-1593). The heterogeneity was revealed by "affinity-labeling" analysis with vanadate plus ADP, which was developed in the previous paper. To see whether this heterogeneity is due to the head-head interaction or two different alkali light chains present in HMM, we carried out similar studies with myosin subfragment-1 (S1) and one of the isozymes, S1(A1), which contains only the alkali light chain 1, and obtained essentially the same results as those previously obtained with HMM. The S1 results are easily explained by the same hypothesis previously used for explaining the HMM results: SH-modified S1 or S1(A1) contains two kinds of active site in a 1:1 ratio with almost the same ATPase activity: one hydrolyzes ATP by a mechanism giving a protein Trp fluorescence enhancement, whereas the other hydrolyzes ATP by another mechanism giving no fluorescence enhancement.

We have examined whether heavy meromyosin (HMM) consists of a single kind of active site by analyzing the changes in the relative MgATPase activity and the relative amplitude of the ATP-induced fluorescence enhancement of the protein when the fraction of HMM "affinity"-labeled by vanadate plus ADP was varied. The analysis is based on a prediction that these two changes should be proportional to each other if myosin consists of a single kind of active site and generates the rate-limiting myosin**product complex emitting enhanced fluorescence. Although the difference between these two changes was very small with native HMM, it was large with HMM in which 5 fast-reactive sulfhydryl-groups per head were pre-modified with thimerosal. The difference indicated the existence of heterogeneous active sites in the SH-modified HMM. The results were best explained in terms of the hypothesis that fifty percent of the active site splits MgATP by a mechanism giving a fluorescence enhancement whereas the other fifty percent splits MgATP by another mechanism giving no fluorescence enhancement. Two possible explanations for the existence of heterogeneous active sites in the SH-modified HMM are discussed. One assumes the pre-existence of some sort of 1:1 heterogeneity in the micro-environment of the active sites and the other, which is considered less likely, assumes the introduction of the heterogeneity as a result of the SH-modification.

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