Effect of Antisera to RuBP-Carboxylase/Oxygenase of TV. tabacum and Spinacia oleracea on the Oxygenase-Function of the Enzyme

In the p resen t p a p e r we d e m o n s tra te th a t the c o n fo rm a tio n a l s ta te o f th e b ifu n c tio n a l enzym e r ib u lo se -1,5 -b isp h o sp h ate carb o x y lase /o x y g en ase is d iffe ren t in th e to b ac co m u ta n t S u /su w hen co m p ared to the w ild type to b acco o r sp inach . T he c o n fo rm a tio n a l s ta te o f th e to b acco m u ­ ta n t enzym e is ch aracterized by th e presence o f a h ig h er n u m b er o f an tig en ic d e te rm in a n ts a c ­ cessible to an tib o d y binding. T h is seem s to be c o rre la te d to a h ig h er oxygenase activ ity in the m u ta n t. T he b ifu nctional enzym e rib u lo se -1,5 -b isp h o sp h a te ca rb o x y lase /o x y g en ase o f the w ild type to b acco Nicotiana tabacum var. Jo h n W illiam ’s B ro ad leaf, o f th e to b ac co m u ta n t S u /su and o f sp in ach (Spinacia oleracea) w as c h arac te rized by c o m p a ra tiv e im m u n o lo g ica l m eth o d s. A lth o u g h the enzym e o f th e to b acco m u ta n t a p p ea rs id en tica l to th e enzym e o f the w ild type, w hen analyzed in im m u n o d iffu sio n tes ts an d im m u n o e le c tro p h o re tica l analyses, it exh ib its a h igher oxygenase activ ity . O n the o th e r h a n d the sp in ach enzym e ex h ib its only p a r ­ tia l sero logical id en tity to the tw o to b acco enzym es. F o r the co m p ara tiv e studies p u re Ig G -frac tio n s w ere p re p a re d from the respec tive an tise ra . R u B P -carboxy lase /oxygenase w as used as a 70% purified enzym e p re p a ra tio n . D e te rm in a tio n o f the an tib o d y b in d in g capacity show ed th a t the enzym es b in d fro m the h o m o lo g o u s an tise ra the h ighest a m o u n t o f an tib o d ies , w hich m ean s th a t the an tise ra reflect the co m p lem en ta ry p ic tu re o f the enzym e stru c tu re . T h e enzym e m olecu les o f N. tabacum var. JW B a n d o f sp inach b ind 9 an tib o d y m olecules each. H ow ever, the b in d in g cap ac ity o f the to b ac co m u ta n t enzym e ex h ib itin g the h ig h er oxygenase activ ity is 30% h igher. M easu rem en t o f the oxygenase fu n c tio n u n d e r th e in fluence o f the h o m o lo g o u s as well as o f the n o n -h o m o lo g o u s an tise ra h as led to the resu lt th a t the oxygenase activ ity o f all enzym es is inh ib ited . H ow ever, it is the degree o f in h ib itio n w hich d iffers. T h e a n tise ru m to the m u ta n t enzym e causes w ith the sp inach as well as w ith th e JW B -enzym e a h ig h er degree o f in h ib itio n th a n th a t p ro d u ced by the h o m o lo g o u s an tise ru m . T h ere fo re , a c o rre la tio n be tw een in h ib ito ry effect b ro u g h t a b o u t by th is an tise ru m an d th e a m o u n t o f a n tib o d ie s b o u n d d oes n o t exist. W hereas the enzym e o f the to b acco wild type b in d s 20% less a n tib o d ie s o u t o f th is an tise ru m its oxygenase activ ity is 60% m o re in h ib ited a n d th e fu n c tio n o f the sp in ach enzym e is 20% stro n g e r in h ib ited a lth o u g h b in d in g o f an tib o d ies from th e an tise ru m to the to b ac co enzym e is 50% low er. T hese o b se rv a tio n s perm it th e co n clusion th a t the an tise ru m to w a rd s the m u ta n t enzym e co n ta in s m o re an tib o d ies w ith a h ig h er b in d in g affin ity to w a rd s reac tive reg io n s o f the ox y ­ genase fu n ction . T h is in tu rn m ean s th a t the s tru c tu re o f th is enzym e o r its c o n fo rm a tio n m u st be d ifferen t in co m p ariso n to the w ild type enzym e.