Characterization of a transglutaminase expressed in human pancreatic adenocarcinoma cells.
A soluble tissue type transglutaminase (TGases; R-glutaminylpeptide:amine gamma-glutamyltransferase, E.C.2.3.2.13) belonging to a group of widely distributed enzymes which catalyze the reaction between a gamma-carboxyamide group of a protein-bound glutamine residue and various amino groups was characterized in cell lines derived from human pancreatic carcinoma. The enzyme activity was measured by incorporation of [3H]putrescine into N,N-dimethylcasein. It showed a strong dependency on Ca2+, which could not be replaced by Mg2+ but was 80% inhibited by 0.7 mM Mg2+ in the presence of optimal Ca2+ concentration (7 mM). The Km-value in regard to putrescine was 2.6 mM. After centrifugation of cell homogenates at 105,000g 95% of the enzyme activity was found in the supernatant indicating that the TGase in pancreatic tumor cells is soluble. This was further substantiated by immunohistochemistry showing a homogeneous cytoplasmic distribution of the TGase in pancreatic tumor cells. Molecular sieve chromatography and Western blot analysis using an antibody against TGase II from human erythrocytes revealed a molecular mass of 80 kDa. In Northern blots with a cDNA of TGase II from mouse macrophages a single transcript approximately 3.4 kbp in size was detected. Polymerase chain reaction analysis using primers for the coding and 3'-non-coding regions showed in each case a single product with the size expected from the human cDNA of TGase II. Taken these data together, we conclude that human pancreatic adenocarcinoma cells express the soluble tissue type TGase II.(ABSTRACT TRUNCATED AT 250 WORDS)
Elsässer
H P
HP
Department of Cell Biology, University of Marburg, Germany.
MacDonald
R
R
Dienst
M
M
Kern
H F
HF
D000230:Adenocarcinoma / Q000201:enzymology*; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011503:Transglutaminases; D008274:Magnesium; D002118:Calcium