Grainger D J, Hesketh T R, Metcalfe J C, Weissberg P L
Department of Biochemistry, University of Cambridge, U.K.
Biochem J. 1991 Jul 1;277 ( Pt 1)(Pt 1):145-51. doi: 10.1042/bj2770145.
Vascular smooth-muscle cells (VSMCs) from rat aortae contained very little non-muscle myosin heavy chain (MHC) immediately after dispersal, and the protein did not accumulate if the cells were held in G0/G1 phase by withholding serum or were held in first S phase by the addition of bromodeoxyuridine (BrdU). However, non-muscle MHC accumulated by greater than 20-fold per cell during first M phase, when over 80% of the cells divided between 48 h and 72 h after addition of serum. Delaying the addition of serum caused a delay in the accumulation of the non-muscle MHC until the cells subsequently entered M phase. If the cells were held in M phase at the metaphase/anaphase boundary by nocadazole, the accumulation of non-muscle myosin still occurred, although division was blocked. When the cells were pulse-labelled with [35S]methionine, it was found that non-muscle MHC was one of the major proteins being made and that its synthesis occurred at similar rates throughout the cell cycle. This implied that the rate of degradation of the protein before first M phase was much faster than in M phase, when the protein accumulated rapidly. This was confirmed by direct measurements of the rate at which [35S]methionine-labelled non-muscle MHC disappeared from the cells, which gave a half-life for the protein of about 8 h before M phase but about 5 days in post-mitotic cells, i.e. an increase of approx. 15-fold. These data are consistent with the hypothesis that there is a mechanism in VSMCs which shortens the half-life of the protein before first M phase and that the accumulation of non-muscle MHC which results from the increase in half-life at first M phase may be necessary for division of these cells.
大鼠主动脉的血管平滑肌细胞（VSMCs）在刚分散后所含的非肌肉肌球蛋白重链（MHC）极少，并且如果通过血清饥饿使细胞停滞在G0/G1期，或者通过添加溴脱氧尿苷（BrdU）使细胞停滞在第一个S期，该蛋白不会积累。然而，在第一个M期，每个细胞中的非肌肉MHC积累超过20倍，此时超过80%的细胞在添加血清后48小时至72小时之间进行分裂。延迟添加血清会导致非肌肉MHC的积累延迟，直到细胞随后进入M期。如果通过诺考达唑使细胞在中期/后期边界停滞在M期，尽管细胞分裂受阻，但非肌肉肌球蛋白的积累仍会发生。当用[35S]甲硫氨酸对细胞进行脉冲标记时，发现非肌肉MHC是主要合成的蛋白质之一，并且其合成在整个细胞周期中以相似的速率进行。这意味着在第一个M期之前该蛋白的降解速率比在M期快得多，在M期该蛋白会迅速积累。通过直接测量[35S]甲硫氨酸标记的非肌肉MHC从细胞中消失的速率证实了这一点，该蛋白在M期之前的半衰期约为8小时，而在有丝分裂后的细胞中约为5天，即增加了约15倍。这些数据与以下假设一致：VSMCs中存在一种机制，该机制会缩短第一个M期之前蛋白质的半衰期，并且第一个M期半衰期增加导致的非肌肉MHC积累可能是这些细胞分裂所必需的。
