High hydrostatic pressure perturbs the interactions between CF(0)F(1) subunits and induces a dual effect on activity.
Chloroplast ATP-synthase is an H(+)/ATP-driven rotary motor in which a hydrophobic multi-subunit assemblage rotates within a hydrophilic stator, and subunit interactions dictate alternate-site catalysis. To explore the relevance of these interactions for catalysis we use hydrostatic pressure to induce conformational changes and/or subunit dissociation, and the resulting changes in the ATPase activity and oligomer structure are evaluated. Under moderate hydrostatic pressure (up to 60-80 MPa), ATPase activity is increased by 1.5-fold. This is not related to an increase in the affinity for ATP, but seems to correlate with an enhanced turnover induced by pressure, and an activation volume for the ATPase reaction of -23.7 ml/mol. Higher pressure (up to 200 MPa) leads to dissociation of the enzyme, as shown by enzyme inactivation, increased binding of 8-anilinonaphthalene-1-sulfonate (ANS) to hydrophobic regions, and labeling of specific Cys residues on the beta and alpha subunits by N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-4-diamine (IAEDANS). Compression-decompression cycles (between 0.1 and 200 MPa) inactivate CF(0)F(1) in a concentration-dependent manner, although after decompression no enzyme subunit is retained on a Sephadex-G-50 centrifuge column or is further labeled by IAEDANS. It is proposed that moderate hydrostatic pressures induce elastic compression of CF(0)F(1), leading to enhanced turnover. High pressure dissociation impairs the contacts needed for rotational catalysis.
Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Al. Bauhinia 400, Cidade Universitária, 21941-590 Rio de Janeiro, RJ, Brazil.
Manuela O
MO
Souza
Tânia B
TB
Creczynski-Pasa
Helena M
HM
Scofano
Peter
P
Gräber
Julio A
JA
Mignaco
D000255:Adenosine Triphosphate / Q000378:metabolism; D025222:Chloroplast Proton-Translocating ATPases / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology; D005453:Fluorescence; D006868:Hydrolysis; D006874:Hydrostatic Pressure; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology; D013194:Staining and Labeling
D021122:Protein Subunits; D000431:Ethanol; D000255:Adenosine Triphosphate; D025222:Chloroplast Proton-Translocating ATPases