Changes in the relative amount of subunits of methionine adenosyltransferase in human lymphocytes upon stimulation with a polyclonal T cell mitogen.

J De La Rosa,H L Legros,A.M Geller,M Kotb

doi:10.1016/s0021-9258(19)50074-6

Abstract

Activation of resting human peripheral blood T lymphocytes by the lectin phytohemagglutinin results in an increase in methionine adenosyltransferase (MAT) activity, accompanied by an increase in the amount of the alpha/alpha' catalytic subunits of the enzyme. In contrast, the amount of the noncatalytic beta subunit remains constant throughout the course of the response. Using both polyclonal antibodies to the holoenzyme and monoclonal antibodies to the alpha/alpha' subunits, we detected a cross-reactive 68-kDa protein, which we refer to as lambda. This protein is present in high abundance in resting T cells but decreases upon cell stimulation, as both MAT activity and the amount of the catalytic alpha/alpha' subunits increase. The decrease in lambda and increase in alpha/alpha' occurs after interleukin-2 production and before DNA synthesis. lambda virtually disappears when the cells are actively dividing. Several continuous T cell lines (HPB-ALL, MOLT-4, and Jurkat) as well as a freshly isolated T cell leukemia (ALL-2) had no detectable lambda. The Km for L-methionine for enzyme from resting peripheral blood mononuclear cells was 19-23 microM, which is 3-8-fold higher than purified MAT from fresh leukemic cells or enzyme from Jurkat cells, both of which have a Km of 3.5-3.8 microM. Kinetic analysis of enzyme activity from activated peripheral blood mononuclear cells suggested the presence of two forms of enzyme catalyzing the synthesis of AdoMet. After separation of lambda from the alpha and beta subunits by hydrophobic chromatography, it was determined that lambda has MAT activity but that it is significantly less active than the form containing the alpha subunit. It therefore appears that in resting T cells MAT is sequestered as a less active form. We hypothesize that lambda is a precursor to the catalytic subunits of human lymphocyte MAT and propose that the transition from lambda to alpha/alpha' may be important in the response of T cells to mitogenic signals.

Highlights

Phocytes by the lectin phytohemagglutinin results in (ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6) cataan increase in methionine adenosyltransferase (MAT) activity, accompanied by an increase in theamount of the ala’ catalytic subunits of the enzyme
Because of the significant metabolic roles of the products of the AdoMet-linked pathways it is not surprising that these reactions play an important role in lymphocyte activation and functional differentiation [1,2,3], ALL, MOLT-4a, nd Jurkat) as well as a freshly isolated and it has been shown that inhibition of AdoMet T cell leukemia (ALL-2) had no detectable X
The K, synthesis blocks T lymphocyteproliferation [5].for L-methionine for enzyme from resting peripheral peripheral blood mononuclear cells (PBMC)stimulated by blood mononuclear cells was 19-23 PM, which is 3-8- phytohemagglutinin (PHA) for 24 h showed a 5-15-fold infold higher than purified MAT from fresh leukemic crease in the amount of AdoMet, as well as an increase in cells or enzyme from Jurkat cells, both of which have AdoMet utilization compared to unstimulated PBMC [6]

Summary

EXPERIMENTAL PROCEDURES

Methods-Aprotinin, antipain, chymotrypsin, leupeptin, pepstatin A, o-phenanthroline, benzamidine, bicinchoninic acid, and PHAwere from Sigma. Preparation of CellExtracts-MAT activity was assayed in extracts prepared from cell pellets by three cycles of freeze-thawing. The enzyme extraction buffer consisted of 50 mM Tris, pH 7.4, 50 mM NaCI, 5 mM MgC12, 4 mM dithiothreitol, and amixture of the followingproteolytic inhibitors: 1mM o-phenanthroline, 10mM benzamidine, 300 pg/ml soy bean trypsin inhibitor, 50 pg/ml aprotinin, 1 pg/ml pepstatin A, 25 pg/ml antipain, 25 pg/ml chymostatin, 25 pg/ ml leupeptin. SDS-PAGE and Western Blotting-The same cell extracts assayed for MAT activity as described above were diluted in loading buffer (60 mM Tris-C1, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, and 5% glycerol), heated in a boiling water bath for 4 min, and analyzed by SDS-PAGE (10% total acrylamide, 2.7% bis-acrylamide [14]). Antibodies to human lymphocyte MAT have been described and characterized previously [9]. The x-ray film was scanned using an LKB 2202 Ultrascan Laser Densitometer

RESULTS

B: Denaturing

DISCUSSION