The effect of “shearing” forces during ultracentrifugation on the membrane-bound polysomes associated with light rough microsomes of MPC-11 cells

Abstract

When microsomes, isolated from MPC-11 cells after nitrogen cavitation of cells in buffer containing 100 mM KCl, were separated into light rough (LR) and smooth (S) fractions by discontinuous gradient centrifugation it was observed that [³H]-choline label and A_{2 6 0 nm} absorbing did not coincide in the LR region of the gradient. This was in contrast to the situation when microsomes were isolated from cells disrupted by nitrogen cavitation at 25 mM KCl. The A_{2 6 0 nm} absorbing material that appeared in gradient fractions (1–5) below the position of LR membranes was found to consist of polysomal material. This material gave a “richer” polysome profile than that released from the LR membranes by addition of detergent. Labelling experiments with [³H]-leucine showed that nascent polypeptides associated with monosomes and polysomes in fraction 1–5 were of shorter length than the corresponding ones in the LR fraction. A mere contamination of LR microsomes by free polysomes appeared most unlikely. The results are consistent with an effect of “shearing” on the membrane-bound polysomes of the LR microsomes under specific experimental conditions. This effect results in the production of a 5′ mRNA fragment (short polypeptide chains) and a 3′ mRNA fragment (long polypeptide chains), the former fragment migrating further down the gradient tube free of LR membranes, whilst the latte remained attached to the LR membranes.