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Titolo:
A novel role of 4,4 '-diisothiocyanatostilbene-2,2 '-disulfonic acid as anactivator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase
Autore:
Santos, FT; Scofano, HM; Barrabin, H; Meyer-Fernandes, JR; Mignaco, JA;
Indirizzi:
Univ Fed Rio de Janeiro, CCS, ICB, Dept Bioquim Med, BR-21941590 Rio De Janeiro, Brazil Univ Fed Rio de Janeiro Rio De Janeiro Brazil BR-21941590 BCeiro, Brazil
Titolo Testata:
BIOCHEMISTRY
fascicolo: 32, volume: 38, anno: 1999,
pagine: 10552 - 10558
SICI:
0006-2960(19990810)38:32<10552:ANRO4'>2.0.ZU;2-I
Fonte:
ISI
Lingua:
ENG
Soggetto:
NEGATIVELY CHARGED PHOSPHOLIPIDS; HUMAN-ERYTHROCYTE-MEMBRANES; CALMODULIN-BINDING DOMAIN; RED-CELL MEMBRANES; CALCIUM-PUMP; CA2+ PUMP; ADENOSINE-TRIPHOSPHATASE; CA-2+ PUMP; CA-2+-PUMPING ATPASE; CIRCULAR-DICHROISM;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
63
Recensione:
Indirizzi per estratti:
Indirizzo: Mignaco, JA Univ Fed Rio de Janeiro, CCS, ICB, Dept Bioquim Med, Cidade Univ, BR-21941590 Rio De Janeiro, Brazil Univ Fed Rio de Janeiro Cidade Univ Rio De Janeiro Brazil BR-21941590 BC
Citazione:
F.T. Santos et al., "A novel role of 4,4 '-diisothiocyanatostilbene-2,2 '-disulfonic acid as anactivator of the phosphatase activity catalyzed by plasma membrane Ca2+-ATPase", BIOCHEM, 38(32), 1999, pp. 10552-10558

Abstract

The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte membrane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a classic inhibitor of anion transport. Enhancement of the phosphatase activity varies from 2- to 6-fold, depending on the Ca2+ and calmodulin concentrations used. Maximumstimulation of the pNPPase activity in ghosts is reached at 4-5 mu M DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate,the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolysis by DIDS is equally effective for both ghosts and purified enzyme, and therefore is independent of its effect as an anion transport inhibitor. Binding of the activator does not change the Ca2+ dependence of the pNPPase activity. Stimulation is partially additive to the activation of the pNPPase activity elicited by calmodulin and appears to involve a strong affinity binding or covalent binding to sulfhydryl groups of the enzyme, since activation is reversed by addition of dithiothreitol but not by washing. The degreeof activation of pNPP hydrolysis is greater at alkaline pH values. DIDS decreases the apparent affinity of the enzyme for pNPP whether in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+ (with 5 muM DIDS the observed K-m shifts from 4.8 +/- 1.4 to 10.1 +/- 2.6, from 3.8 /- 0.4 to 7.0 +/- 0.8, and from 9.3 +/- 0.7 to 15.5 +/- 1.1 mM, respectively). However, the pNPPase rate is always increased las above, from 3.6 +/- 0.6 to 11.2 +/- 1.7, from 4.4 +/- 0.5 to 11.4 +/- 0.9, and from 2.6 +/- 0.6to 18.6 +/- 3.9 nmol mg(-1) min(-1), in the presence of Ca2+ alone or Ca2and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the pNPPase activity in the absence of Ca2+5, both in the presence and in the absence of DIDS. Therefore, kinetic evidence indicates that DIDS does more than shift the enzyme to the E-2 conformation. We propose that the transition from E-2 to E-1 is decreased and a new enzyme conformer, denoted E-2*, isaccumulated in the presence of DIDS.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 10/04/20 alle ore 15:16:53