P-E4-11 Subunit structure analysis of H+-PPase by radiation inactivation and hydrostatic pressure studies

E4 ATPases and proton pumps P-E4-10 ACTIVE CATgcuY GRELL E,’ DOLUDDA M,’ RUE H,’ SCHICK E,’ LEWITZKI E,’ TBALER A,’ BUGNON P, MERBACH A E,2MUTZ M,3 MART1 E.3

PE4-09 COWER, H.G.L. & CHCLCOTT,T.C. UNESCO Centre for Membrane Scienceand Technology and w of Biophysics, University of NSW, Sydney2652, Austraha

‘Max-Planck-Inst. Biiphys., Fraaldbi (D). %st. Ana@ Chem., Lmsanne (CH). Ciba-Geigy AG, Basel (CH)

Purpose: Thermodynamicand kinetic studiesare carried out to characterizeoverall and single cation binding equilibria and to elucidatethe detailed mechanismof binding to Na,K-ATPase. Methods: Resultsare obtainedby spectrofluorometric and calorimetric titrations as well as by ambient- and high-pressurestopped-flow kinetics employing highly purified Na,K-ATPaseand its fluorescentFITC-derivative. Results: Equilibrium constantsand enthalpies,rate constantsand activation energiesaswell as vohune changesand activation volumesare determined. Non-selectiveand selectivecation binding to Na,K -ATPaseis very fast but can be distinguishedon the basisof different stoichiometriesand different subsequent,slow conformational rearrrangements. For selectiveK’ binding, the volume changeof the conformational transition, assignedto cation occlusion, is comparativelysmall but the activation volumes are positive and very high. This could be indicative of a substantialrearraugementwithin the transmembraneregion of the protein. P-E4-11

P-E4-12

SUBUNIT STRUCTURE ANALYSIS OF H+-PPaseBY RADIATION INACTIVATION AND HYDROSTATIC PRESSURE‘sTuI)1;ES SHIH SHENG JIANG, CBI MENG TZENG, SU JING YANG, RONG LONG PAN Inst. of Radiation

STRUCTURAL MAPPING OF VACUOLAR H+-PYR~P~~~SP~TASE SU JJ.NGYANG, SHIH SBENGAANG, RONG LONG PAN Inst.of RadiationRioiegyandDept.of Nuclear Science, College of Nuclear Science,Natl. Tsing Hua Univ. (Taiwan, ROC)

Biology and Dept. of Nuclear Science,Collage of Nuclear Science,Natl. Tsing Hua Univ. (Taiwan, ROC)

Purpose: The group specificchemicalprobes were used to identify the essentialamino acid residues and to investigatethe rekuionshipbetween structure and function of vacuolarH’-PPase. Methods: The vacuolar@-PPasewas purified from etiolated mung bean seedlings.Chemical modification, kinetic analysis,and spectroscopic studieswere used to determinethe csseutialamino acid residuesin the active site of I-l+-PPase. Results: AR chemical probesemployedresulted in the marked decline in enzymaticactivitiesand its associatedIT-translocation. Kinetic analysisand protection study indicated that thesechemicals probably attacked the catalyticsite of vacuolarH’PPase. Conclusions: This study suggestedthat at least one essentialcarboxylateres&e, one essentialtyrosine residue, and one essentiallysineresidueare involved in the enzymaticactivity. The exsctsites of these essentialresiduesare to be identified.

Purpose: In order to examinethe subunit structure of vacuolar H+-PPase,radiation inactivationwas used to determine the fUnctionalmassof @-PPase under variousenvironments.Also, the effectsof pressureon its conformation was investigated. Methods: HI-PPasewere irradiated with %o or exposedto various hydrostaticpressuresin an Aminco French PressCell. The functional massof radiation inactivationwas obtained by the target size analysis. Results: Purified I-l+-PPase (140 kDa) is a dimer containing a singlekind polypeptide (73 kDa). Target size analysis yield functionalmasses of 141.5 and 158.4 kDa for enxymaticactivity and its associatedproton translocationrespectively.In addition, high pressurecausedthe dissociationof H’-PPasefrom dimeric into monomericform. Coaclusions: Both studies confirmedthe in situ dimeric nature of membrane-bound H’-PPaseof plant vacuoles.A wo&h@medel is pmposedto elucidateits structureunder variouscoaditions. 166