Synthesis of a radioactive azido derivative of thapsigargin and photolabeling of the sarcoplasmic reticulum ATPase

被引：22

作者：

Hua, SM ^{[1
]}

Inesi, G ^{[1
]}

机构：

[1] UNIV MARYLAND, SCH MED, DEPT BIOCHEM & MOL BIOL, BALTIMORE, MD 21201 USA

来源：

BIOCHEMISTRY | 1997年 / 36卷 / 39期

关键词：

D O I：

10.1021/bi970105n

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

A thapsigargin C8-derivative (ZTG) was synthesized by acylating debutanoylthapsigargin with 4-azido[carboxyl-C-14]benzoic acid. ZTG retains the inhibitory activity of thapsigargin (TG) with respect to the Ca2+ ATPase of sarcoplasmic reticulum (SR). Covalent ATPase labeling was obtained by photoactivation of the ZTG azido moiety under conditions optimized to reduce nonspecific association of ZTG with SR vesicles and to approximate a matching ZTG:ATPase stoichiometry. Specific photolabeling of the Ca2+ ATPase with ZTG was obtained with 30% efficiency and was competitively inhibited by TG. Analysis of the labeled protein and its proteolytic fragments demonstrates that the ZTG label is associated covalently with the membrane-bound portion of tryptic subfragment Al, which spans the sequence between Leu253 and Arg324 and includes segments of S3 and S4 in the stalk, the M3 and M4 transmembrane helices, and the intervening lumenal loop. This finding is in agreement with previous spectroscopic observations and mutational analysis.

引用

页码：11865 / 11872

页数：8

共 29 条

[1]

CAMPBELL AM, 1991, J BIOL CHEM, V266, P16050

[2] INTERPRETATION OF THE NMR AND CIRCULAR DICHROIC DATA OF THE SESQUITERPENE LACTONE THAPSIGARGIN [J].

CHRISTENSEN, SB .

ACTA CHEMICA SCANDINAVICA SERIES B-ORGANIC CHEMISTRY AND BIOCHEMISTRY, 1988, 42 (09) :623-628

[3]

CHRISTENSEN SB, 1982, J ORG CHEM, V47, P649

[4] DERIVATIVES OF THAPSIGARGIN AS PROBES OF ITS BINDING-SITE ON ENDOPLASMIC-RETICULUM CA2+ ATPASE - STEREOSELECTIVITY AND IMPORTANT FUNCTIONAL-GROUPS [J].

CHRISTENSEN, SB ;

ANDERSEN, A ;

POULSEN, JCJ ;

TREIMAN, M .

FEBS LETTERS, 1993, 335 (03) :345-348

[5] LOCATION OF HIGH-AFFINITY CA-2+-BINDING SITES WITHIN THE PREDICTED TRANSMEMBRANE DOMAIN OF THE SARCOPLASMIC-RETICULUM CA-2+-ATPASE [J].

CLARKE, DM ;

LOO, TW ;

INESI, G ;

MACLENNAN, DH .

NATURE, 1989, 339 (6224) :476-478

[6]

DEMEIS L, 1979, ANNU REV BIOCHEM, V48, P275

[7] PHOSPHOLIPID ORIENTATION IN SARCOPLASMIC MEMBRANES - SPIN-LABEL ESR AND PROTON NMR STUDIES [J].

ELETR, S ;

INESI, G .

BIOCHIMICA ET BIOPHYSICA ACTA, 1972, 282 (SEP1) :174-&

[8]

HORGAN DJ, 1972, ANAL BIOCHEM, V48, P147, DOI 10.1016/0003-2697(72)90178-9

[9] SYNTHESIS AND INTERACTION OF FLUORESCENT THAPSIGARGIN DERIVATIVES WITH THE SARCOPLASMIC-RETICULUM ATPASE MEMBRANE-BOUND REGION [J].

HUA, SM ;

MALAK, H ;

LAKOWICZ, JR ;

INESI, G .

BIOCHEMISTRY, 1995, 34 (15) :5137-5142

[10] TRYPTIC CLEAVAGE OF SARCOPLASMIC-RETICULUM PROTEIN [J].

INESI, G ;

SCALES, D .

BIOCHEMISTRY, 1974, 13 (16) :3298-3306

← 1 2 3 →