A CROMOGLYCATE BINDING-PROTEIN FROM RAT MAST-CELLS OF A LEUKEMIA LINE IS A NUCLEOSIDE DIPHOSPHATE KINASE

作者：HEMMERICH S; YARDEN Y; PECHT I

来源：Biochemistry, 1992, 31(19): 4574-4579.

DOI：10.1021/bi00134a006

Recently, we have shown that a membrane-permeant derivative of the antiasthmatic drug cromoglycate (CG) effectively inhibits the Fc(epsilon)-receptor-mediated secretory response of rat mucosal mast cells (line RBL-2H3) at a stage preceding the transient rise in the cytoplasmic free calcium concentration [Hemmerich, S., Sijpkins, D., & Pecht, I. (1991) Biochemistry 30, 1523-15321. In contrast to cromoglycate itself, which is membrane impermeant and ineffective in these cells, its bis-acetoxymethyl ester derivative (CG/AM) can diffuse across the plasma membrane into the cytosol, where it is hydrolyzed into the impermeant CG dianion, which presumably may interact with intracellular components involved in the Fc(epsilon)R signal transduction pathway. In order to identify cytosolic components involved in the stimulus-secretion coupling that interact with this drug, we coupled CG to an insoluble matrix. This matrix was indeed effective in the affinity isolation from RBL cells of a cytosolic protein that exhibits an apparent molecular mass of 18 kDa on reducing SDS gels. This protein was purified to homogeneity and then fragmented, and the amino acid sequence of three resultant peptides was determined. Using the corresponding synthetic oligonucleotides, we cloned and sequenced a cDNA that encodes the full-length 18-kDa polypeptide (p18). The protein sequence deduced from this cDNA is identical to that of rat nucleoside diphosphate kinase [Kimura, N., Shimada, N., Nomura, K., & Watanabe, K. (1990) J. Biol. Chem. 265,15744-157491 and highly homologous (88%) to the human NM23 gene product whose expression is associated with reduced metastatic potential, as well as with the Drosophila awd gene product (77% sequence identity). The p18 isolated from RBL cells was indeed shown to catalyze phosphate transfer from nucleoside triphosphates to nucleoside diphosphates. This activity was inhibited by cromoglycate (I50 almost-equal-to 2 mM). The amino acid sequence of p18 and its enzymatic activity indicate that this cromoglycate binding protein is a nucleoside 5'-diphosphate kinase, which, through control of the cytoplasmic pool of nucleoside triphosphates (e.g., GTP), may affect the transduction of the Fc(epsilon)-receptor-mediated signal.

• 出版日期1992-5-19