2 DISTINCT CLASSES OF RAT INTESTINAL MUCOSAL ENZYMES INCORPORATING PUTRESCINE INTO PROTEIN

Tissue-transglutaminase (t-TGase) is a family of calcium-dependent enz ymes. A Ca2+-independent soluble enzyme, in addition to t-TGase, capab le of incorporating polyamines into proteins was demonstrated in rat i ntestinal mucosa, The Ca2+-independent enzyme was stimulated 2- to 5-f old by Fe2+ and Co2+ ions but inhibited by Cu2+ and Zn2+ ions, The Ca2 +-stimulated t-TGase activity was inhibited by divalent ions in the fo llowing order: Zn2+, Fe2+ > Co2+ > Cu2+ The opposite effects of EGTA, Fe2+ and Co2+ on these two enzyme activities indicate that they are tw o distinct classes of enzymes. Competition studies demonstrated differ ential preferences of the two enzymes for substrates, The Ca2+-depende nt enzyme preferred putrescine, monodansylcadaverine > cadaverine, spe rmidine, spermine > 1,10-diaminodecane > triethylbutylamine. On the ot her hand, the Ca2+-independent enzyme preferred putrescine > cadaverin e > spermine, 1,10-diaminodecane > spermidine > monodansylcadaverine > triethylbutylamine. Further studies with divalent ions excluded the p ossible association of this novel Ca2+-independent enzyme with diamine oxidase, Finally, the Ca2+-independent enzyme had a higher affinity f or putrescine (K-m = 0.02 mM) than did Ca2+-dependent t-TGase (0.2 mM) , As judged by gel filtration on HiPrep Sephacryl 200 column, the Ca2-independent enzyme had a molecular weight of similar to 48 kDa, the i ntestinal Ca2+-dependent t-TGase was about 188 kDa while that of testi cular t-TGase was about 96 kDa, In conclusion, the Ca2+-independent en zyme is stimulated by cobalt or ferric ions, and selectively incorpora tes aliphatic diamines or polyamines with symmetric amino groups. The observed Ca2+ independent enzyme activity is not related to diamine ox idase or its products. With a 10 times greater affinity for putrescine , the calcium-independent, 48-kDa intestinal enzyme may mediate polyam ine function better than calcium dependent, 188-kDa intestinal tissue transglutaminase in the intestinal mucosa, (C) 1998 Federation of Euro pean Biochemical Societies.