Molecular and Cellular Biochemistry 75:33-42 (1987) © Martinus NijhhoffPublishers, Boston - Printed in the Netherlands 33 Original Article Increase in the levels of activity of polyadenylic acid-metabolizing enzymes following phytohaemagglutinin stimulation of human lymphocytes Nelly C. Courtis 1, Theoni T. Trangas and Chris M. Tsiapalis Department of Biochemistry, "G. Papanikolaou" Research Center, Hellenic Anticancer Institute, Athens 115 22, Greece (1address for offprints) Received 25 November 1986 Keywords: polyadenylic acid, phytohaemagglutinin, peripheral lymphocytes Abstract Increased levels of soluble activity of all three enzymes involved in polyadenylic acid metabolism were meas- ured in PHA-stimulated versus normal lymphocytes. Poly(A)-polymerase and poly(A)-exonuclease values in- creased significantly (from 25.7 + 4.2 (S.E.M.) to 53.5 + 10.6 (S.E.M.), and from 334.6 + 33.2 (S.E.M.) to 653.2 _+ 53.4 (S.E.M.) respectively), while a moderate increase was observed in poly(A)-endonuclease (from 299.2 + 33.8 (S.E.M.) to 403.0 + 77.1 (S.E.M.). The above differences persisted after two fractionations of the crude cell extracts by ion exchange chromatography and molecular sieving, and could not be attributed to the competitive action of all three enzymes in the untreated extracts. Fractionation of the extracts of rest- ing and stimulated cells on Sephadex G-75 revealed two molecular forms of poly(A)-polymerase activity. Abbreviations: Polyadenylic acid, poly(A) or An; oligoadenylic acid, oligo(A) or A10; polycytidylic acid, poly(C); polyuridylic acid, poly(U); polyguanylic acid, poly(G); polydeoxyadenylic acid poly(dA); ethylenediamine tetraacetate, EDTA: phytohaemagglutinin, PHA: phosphate buffered saline, PBS: nonidet-40; NP-40; potassium phosphate buffer, KPi buffer. Introduction PHA permits the study of lymphocyte activation in vitro. When added to resting lymphocytes in cul- ture it interacts with surface receptors and induces cells to undergo dramatic biochemical and physio- logical changes, culminating in DNA replication and cell division. PHA, a T-cell-specific lectin, has been broadly used to investigate changes in the morphology of cells (1, 2, 3) as well as DNA replication (2), RNA (4) and protein synthesis in general (4). PHA stimu- lation has been found to cause the loss of a labile class of poly(A) (+)mRNA of half-life less than 20 min of resting lymphocytes. In the activated cells only the stable class of 50 hr half-life prevailed (5, 6). The polyadenylation of RNA reached a three-fold increase after 40 hr of lectin stimulation, though the estimated maximum length of the poly(A) tail was reduced from 240 residues in rest- ing cells to 220 in activated (7). Translation in cell- free system of the whole mRNA population of rest- ing and activated lymphocytes revealed that the same qualitative protein pattern existed in both categories of lymphocytes, while the quantity differed upon stimulation, indicating that transla-