REGULATION OF CALMODULIN-BINDING TO THE ATP EXTRACTABLE 110 KDA PROTEIN (MYOSIN-I) FROM CHICKEN DUODENAL BRUSH-BORDER BY 1,25-(OH)2D3

In earlier studies we observed that the active vitamin D metabolite 1, 25-(OH)2D3 increased the calmodulin co tent of purified duodenal brush -border membrane vesicles where it bound principally to the 110 kDa pr otein myosin I. In this study we further evaluated the regulation of c almodulin binding to ATP releasable myosin 1. 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 extracta ble myosin I from BB resulted in an 1.6-fold increase of calmodulin bi nding to the 110 kDa band after treatment with 1,25-(OH)2D3. In contra st to BB, ATP extraction of myosin I from purified BBMV required alame thicin for ATP entry. As for BB extracts, calmodulin binding to the 11 0 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 myo sin I by 1,25-(OH)2D3. To see whether 1,25-(OH)2D3 increased the intri nsic affinity of calmodulin binding to myosin I, the ATP extractable m yosin I from BB was purified from rachitic chicks treated with 1,25-(O H)2D3 or vehicle. In contrast to ATP extracts of BB or BBMV, calmoduli n binding to the purified myosin I was not different between preparati ons from 1,25-(OH)2D3- or vehicle-treated chicks. We conclude that 1,2 5-(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 1. Phosphorylation of brush-border membrane proteins in viv o was performed by incubation of [P-32]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-P-32]ATP for 1 min. Incubation experiments in vivo and in vitro in fact resulte d in phosphorylation of several proteins including 110 kDa proteins. H owever, there was no specific effect of 1,25-(OH)2D3 on phosphorylatio n 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-(O H)2D3 stimulated calmodulin binding to ATP extractable brush-border me mbrane myosin I and 1,25-(OH)2D3 stimulated changes of calcium uptake across the brush-border membrane.