Inhibition of the assembly and secretion of procollagen by incorporation of a threonine analogue, hydroxynorvaline.
Hydroxynorvaline (DL-alpha-amino-beta-hydroxyvaleric acid) was shown to competitively inhibit the activation of threonine and valine when tested with tRNA and synthetases prepared from whole chick embryos. However, the hydroxynorvaline was transferred only to threonyl-tRNA and not valyl-tRNA. The hydroxynorvaline had no effect when tested with other amino acids. The Km for threonine was 25 muM and the Ki for hydroxynorvaline was 181 muM. When fibroblasts from embryonic chick tendons were incubated with [3H]threonine and increasing concentrations of hydroxynorvaline, there was a progressive decrease in the incorporation of [3H]threonine so that 1 mM hydroxynorvaline the incorporation into nondialyzable protein was 26% of the control value. A much smaller decrease in the incorporation of other radioactive amino acids was observed. When the cells were incubated hith [14C]proline and 1 mM hydroxynorvaline, the labeled procollagen containing hydroxynorvaline accumulated intracellularly and very little was secreted. Control experiments demonstrated that free hydroxynorvaline did not inhibit the secretion of unsubstituted procollagen. Although the individual pro alpha chains containing hydroxynorvaline were of normal molecular weight (125,000) and hydroxyproline content, only about 50% of this intracellularly retained procollagen was triple helical within the cell as 37 degrees as measured by sensitivity to pepsin digestion. Also only approximately 50% of the pro alpha chains were disulfide-linked to form triple stranded molecules as compared to greater than 85% linkage in unsubstituted procollagen. We postulate that incorporation of hydroxynorvaline alters the conformation of the propeptide extension sufficiently so that: (a) normal assembly of disulfide-linked, triple helical molecules is reduced and (b) assembled triple helical molecules are not properly recognized by the secretory mechanism.
D000604:Amino Acyl-tRNA Synthetases / Q000037:antagonists & inhibitors; D000818:Animals; D002642:Chick Embryo; D003094:Collagen / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D007700:Kinetics; D011498:Protein Precursors / Q000378:metabolism*; D013696:Temperature; D013912:Threonine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D013914:Threonine-tRNA Ligase / Q000037:antagonists & inhibitors; D046249:Transfer RNA Aminoacylation / Q000187:drug effects; D014633:Valine / Q000378:metabolism
D011498:Protein Precursors; D013912:Threonine; D003094:Collagen; D000604:Amino Acyl-tRNA Synthetases; D013914:Threonine-tRNA Ligase; D014633:Valine