MULTIPLE UBIQUITIN C-TERMINAL HYDROLASES FROM CHICK SKELETAL-MUSCLE

A new method for assaying ubiquitin C-terminal hydrolases was develope d using a I-125-labeled ubiquitin-alpha NH-MHISPPEPESEEEEEHYC as subst rate, Since the peptide portion was almost exclusively radiolabeled, t he enzymes could be assayed directly by simple measurement of the radi oactivity released into acid-soluble products, Using this assay protoc ol, we identified at least 10 ubiquitin C-terminal hydrolase activitie s from the extract of chick skeletal muscle, which were tentatively na med UCHs 1 through 10, Of these, UCH-B was purified to apparent homoge neity, Purified UCH-6 behaved as a dimer of 27-kDa subunits, The appar ent molecular masses of the other partially purified UCHs ranged hom 3 5 to 810 kDa as determined under a nondenaturing condition, Muscle UCH s, except UCH-1, were activated dramatically by poly-L-Lys but with an unknown mechanism. Ah of the UCHs were sensitive to inhibition by sul fhydryl-blocking agents such as iodoacetamide. In addition, all of the UCHs were capable of releasing free ubiquitin from a ubiquitin-alpha NH-carboxyl extension protein of 80 amino acids and from ubiquitin-alp ha NH-dihydrofolate reductase, Five of the enzymes, UCHs 1 through 5, were also capable of generating free ubiquitin from poly-His-tagged di ubiquitin. In addition, UCH-1 and UCH-7 could remove ubiquitin that ha d been ligated covalently by an isopeptide linkage to a ubiquitin(RGA) -alpha NH-peptide, the peptide portion of which consists of the 20 ami no acids of the calmodulin binding domain of myosin light chain kinase , These results suggest that the 10 UCH activities isolated from chick skeletal muscle appear to be distinct from each other at least in the ir chromatographic behavior, size, and substrate specificity.