Regulation of calmodulin binding to the ATP extractable 110 kDa protein (myosin I) from chicken duodenal brush border by 1,25-(OH)2D3

Abstract

In earlier studies we observed that the active vitamin D metabolite 1,25-(OH)2D3 increased the calmodulin content of purified duodenal brush-border membrane vesicles where it bound principally to the 110 kDa protein myosin I. In this study we further evaluated the regulation of calmodulin binding to ATP releasable myosin I. Whole brush borders (BB) or purified brush-border membrane vesicles (BBMV) were prepared from duodena of vitamin D-deficient rachitic chicks treated 12-18 h before killing with either 625 pmol 1,25-(OH)2D3 or vehicle. The ATP extractable myosin I from BB resulted in an 1.6-fold increase of calmodulin binding to the 110 kDa band after treatment with 1,25-(OH)2D3. In contrast to BB, ATP extraction of myosin I from purified BBMV required alamethicin for ATP entry. As for BB extracts, calmodulin binding to the 110 kDa band in BBMV extracts was also increased about 2.4-fold by 1,25-(OH)2D3. It was concluded that both intact BB and purified BBMV showed the same type of increase in calmodulin binding to ATP releasable myosin I by 1,25-(OH)2D3. To see whether 1,25-(OH)2D3 increased the intrinsic affinity of calmodulin binding to myosin I, the ATP extractable myosin I from BB was purified from rachitic chicks treated with 1,25-(OH)2D3 or vehicle. In contrast to ATP extracts of BB or BBMV, calmodulin binding to the purified myosin I was not different between preparations from 1,25-(OH)2D3- or vehicle-treated chicks. We conclude that 1,25-(OH)2D3 does not change the affinity of calmodulin binding to myosin I but increases the amount of myosin I in the membrane or alters its ATP releasability. It was further investigated whether phosphorylation is involved in these 1,25-(OH)2D3 dependent posttranslational changes of myosin I. Phosphorylation of brush-border membrane proteins in vivo was performed by incubation of [32P]P(i) in the lumen of a ligated duodenal loop in situ for 15 min. Brush-border membrane proteins were phosphorylated in vitro by incubating BB or BBMV with [gamma-32P]ATP for 1 min. Incubation experiments in vivo and in vitro in fact resulted in phosphorylation of several proteins including 110 kDa proteins. However, there was no specific effect of 1,25-(OH)2D3 on phosphorylation of 110 kDa proteins. We conclude that the effects of 1,25-(OH)2D3 on protein phosphorylation are minimal and not likely to explain 1,25-(OH)2D3 stimulated calmodulin binding to ATP extractable brush-border membrane myosin I and 1,25-(OH)2D3 stimulated changes of calcium uptake across the brush-border membrane.