Mr M V RAMANUJAM

Topics: Enzyme, Protein, Bacillus Pages: 1 (300 words) Published: May 29, 2014
Transglutaminase from Bacillus subtilis 168 cells.

FASEB Journal 4, A 2321 (1990).

INTRACELLULAR TRANSGLUTAMINASE (EC 2.3.2.13) IN A PROCARYOTE: EVIDENCE FROM VEGETATIVE AND SPORULATING CELLS OF BACILLUS SUBTILIS 168. M.V. Ramanujam and J.H. Hageman, New Mexico State University, Las Cruces, NM 88003.

We showed earlier that not only is Ca²+ required for sporulation of B. subtilis cells but it is also necessary to carry out normal rates of protein degradation during sporulation. Our previous work and that of others have suggested that proteolytic cleavage may not be the rate limiting step in bulk protein degradation, implying marking or post translational modification of the proteins might precede the proteinases action. [Folia Microbiol. 32, 465-480 (1987)]. As transglutaminase from eucaryotic systems were known to require Ca²+ and to bring about post translational modifications of native proteins, we screened for and found transglutaminase activity in cell free extracts of cells of B. subtilis growing and sporulating in chemically defined medium. Both soluble and membrane fractions contained a heat-labile activity which incorporated (14C) putrescine into N, N1 dimethylated casein and into endogenous cellular protein. The enzyme remained in the supernatant fraction after addition of (NH2)4SO4 to 70% saturation, yielding over 200-fold purification and 2-fold increase in total units. This partially purified activity incorporated putrescine into dimethylated casein linearly with time and added enzyme. Activity was optimal above pH 9.5; in CHES buffer at pH 9.5 the transglutaminase was completely inhibited by 10 mM EGTA, by Ca²+ above 5mM, by dithiothreitol (64% at 3 mM) and by cystamine (66% at 50μM). The apparent Km for dimethylated casein was 5 mg/ml. Spermidine, spermine and dansyl cadaverine competed with putrescine for incorporation into dimethylated casein. Thus the transglutaminase of B. subtilis cells might be a candidate for...
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