[show abstract][hide abstract] ABSTRACT: Tissue-transglutaminase (t-TGase) is a family of calcium-dependent enzymes. A Ca2+-independent soluble enzyme, in addition to t-TGase, capable of incorporating polyamines into proteins was demonstrated in rat intestinal mucosa. The Ca2+-independent enzyme was stimulated 2- to 5-fold 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 following order: Zn2+, Fe2+ >Co2+ > Cu2+. The opposite effects of EGTA, Fe2+ and Co2+ on these two enzyme activities indicate that they are two distinct classes of enzymes. Competition studies demonstrated differential preferences of the two enzymes for substrates. The Ca2+-dependent enzyme preferred putrescine, monodansylcadaverine > cadaverine, spermidine, spermine > 1,10-diaminodecane > triethylbutylamine. On the other hand, the Ca2+-independent enzyme preferred putrescine > cadaverine > spermine, I,10-diaminodecane > spermidine > monodansylcadaverine > triethylbutylamine. Further studies with divalent ions excluded the possible association of this novel Ca2+-independent enzyme with diamine oxidase. Finally, the Ca2+-independent enzyme had a higher affinity for putrescine (Km = 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 approximately 48 kDa, the intestinal Ca2+-dependent t-TGase was about 188 kDa while that of testicular t-TGase was about 96 kDa. In conclusion, the Ca2+-independent enzyme is stimulated by cobalt or ferric ions, and selectively incorporates aliphatic diamines or polyamines with symmetric amino groups. The observed Ca2+-independent enzyme activity is not related to diamine oxidase or its products. With a 10 times greater affinity for putrescine, the calcium-independent, 48-kDa intestinal enzyme may mediate polyamine function better than calcium dependent, 188-kDa intestinal tissue transglutaminase in the intestinal mucosa.
[show abstract][hide abstract] ABSTRACT: A Ca(++)-independent enzyme capable of incorporating [3H]-putrescine into proteins was detected in the rat intestine mucosa. The Ca(++)-independent incorporation of [3H]-putrescine into proteins was temperature-, pH-, time-, and dose-dependent. However, this enzyme was absent in the gastric mucosa. Similar to testicular Ca(++)-dependent transglutaminase, the optimal pH of intestinal Ca(++)-independent enzyme was 9.0. At 10(-5) M or less putrescine concentrations, the Ca(++)-independent enzyme in an intestinal cytosol preparation showed a greater activity than did the Ca(++)-dependent transglutaminase. However, at higher putrescine concentrations, the latter showed a greater activity than did the former. Both the intestinal Ca(++)-dependent and independent enzymes were inhibited by cystamine, thermal labile at 50 degrees C and precipitated by 30 to 50% saturation of ammonium sulfate. The fact that these two enzymes shared many similar characteristics, with the exceptions of Ca(++)-requirement, suggests that they may have similar active site and intrinsic molecular function(s).
Biochemical and Biophysical Research Communications 03/1998; 244(1):161-6. · 2.41 Impact Factor