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Titolo:
PURIFICATION AND CHARACTERIZATION OF HUMAN ZAP-70 PROTEIN-TYROSINE KINASE FROM A BACULOVIRUS EXPRESSION SYSTEM
Autore:
ISAKOV N; WANGE RL; WATTS JD; AEBERSOLD R; SAMELSON LE;
Indirizzi:
NICHHD,CELL BIOL & METAB BRANCH,NIH BETHESDA MD 20892 NICHHD,CELL BIOL & METAB BRANCH,NIH BETHESDA MD 20892 BEN GURION UNIV NEGEV,DEPT MICROBIOL & IMMUNOL IL-84105 BEER SHEVA ISRAEL BEN GURION UNIV NEGEV,CANC RES CTR IL-84105 BEER SHEVA ISRAEL UNIV WASHINGTON,DEPT MOLEC BIOTECHNOL SEATTLE WA 98195
Titolo Testata:
The Journal of biological chemistry
fascicolo: 26, volume: 271, anno: 1996,
pagine: 15753 - 15761
SICI:
0021-9258(1996)271:26<15753:PACOHZ>2.0.ZU;2-S
Fonte:
ISI
Lingua:
ENG
Soggetto:
CELL ANTIGEN RECEPTOR; T-CELL; ZETA-CHAIN; CYTOPLASMIC DOMAIN; INITIAL CHARACTERIZATION; HUMAN PLATELETS; SH2 DOMAINS; ACTIVATION; P56LCK; ASSOCIATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
66
Recensione:
Indirizzi per estratti:
Citazione:
N. Isakov et al., "PURIFICATION AND CHARACTERIZATION OF HUMAN ZAP-70 PROTEIN-TYROSINE KINASE FROM A BACULOVIRUS EXPRESSION SYSTEM", The Journal of biological chemistry, 271(26), 1996, pp. 15753-15761

Abstract

The ZAP-70 protein tyrosine kinase is essential for T cell antigen receptor (TCR)-mediated signaling. The absence of ZAP-70 results in impaired differentiation of T cells and a lack of responsiveness to antigenic stimulation. In order to study the characteristics of ZAP-70 in vitro, we overexpressed an epitopically tagged human ZAP-70 in a recombinant baculovirus expression system and purified it by column chromatography. The kinase activity of purified, recombinant ZAP-70 required cation and exhibited a strong preference for Mn2+ over Mg2+. The apparent K-m of ZAP-70 for ATP was similar to 3.0 mu M. The activity of the recombinant ZAP-70, unlike that of the homologous protein tyrosine kinase, Syk, was not affected by binding of TCR-derived tyrosine phosphorylated immunoreceptor tyrosine-based activation motif peptides. Severalproteins were tested as potential in vitro substrates of ZAP-70. Onlyalpha-tubulin and the cytoplasmic fragment of human erythrocyte band 3 (cfb3), which have a region of sequence identity at the phosphorylation site, proved to be good substrates, exhibiting K-m values of similar to 3.3 and similar to 2.5 mu M respectively ([ATP] = 50 mu M). alpha- and beta-Casein were poor substrates for ZAP-70, and no activity toward enolase, myelin basic protein, calmodulin, histone proteins, or angiotensin could be detected. In contrast to the T cell protein tyrosine kinase, Lck, ZAP-70 did not phosphorylate the cytoplasmic portion of the TCR zeta chain or short peptides corresponding to the CD3 epsilon or the TCR zeta immunoreceptor tyrosine-based activation motifs. Ourstudies suggest that ZAP-70 exhibits a high degree of substrate specificity.

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Documento generato il 30/11/20 alle ore 19:49:32