Phorbol ester-induced membrane proteins in chronic leukemic B-lymphocytes. Candidate proteins for the L-system amino acid transporter

Abstract

Chronic lymphocytic leukemia (CLL) B-lymphocytes have markedly diminished membrane L-system amino acid transport as compared with normal mature B- and T-lymphocytes. L-system functional recovery is induced in CLL B-cells by the maturational agent, 12-O-tetradecanoylphorbol-13-acetate (TPA). The studies reported here extend the analysis of CLL B-cell maturation by comparing membrane protein expression in untreated and TPA-treated CLL B-cells, with the identification of candidate proteins for the L-system transporter. Cell membrane proteins of resting and TPA-treated CLL B-lymphocytes were studied using ultrahigh resolution giant two-dimensional gel electrophoresis. Cellular proteins were metabolically labeled with [35S]methionine, and, in separate experiments, membrane proteins were photoaffinity labeled with [125I] iodoazidophenylalanine, an amino acid transporter by the L-system and which binds at or near the L-system transport carrier. In a partially purified membrane preparation, approximately 1400 proteins were identified by metabolic labeling. Following TPA treatment for 17 h, 14 new metabolically labeled membrane proteins were identified, and five of these also were labeled by the L-system photoprobe. Photolabeling of four of these proteins was inhibited by an excess of the L-system prototype amino acid, 2-aminobicyclo(2.2.1)heptane-2-carboxylic acid. Given these labeling characteristics, one or more of these four proteins may be related to the L-system amino acid transport carrier.