LaY De1> Botánica y F. Veg. 10: 49-52 (1979) Madrid, 10 febrero 1079 ¡

EFFECT OF L-USNIC ACID ON A THERMICALLYDENATURED UREASE

por

BLANCA CIFUENJESand O VWENIIY

1 X[ R O O U c T 1 0 N

Usi>ic acids are powerful cm]zy>]e inactivators, being demonstratedfor DNAase (1). glutamate dehydrogenase(2) and urease (a). Thisft>>]ction isacommon cl]aracteristic of phenolics (4), cat>5i1]g aggrega—

tío>] sUdes wl]ici> increase >nolecular -weigbt of protcíns. iZece>lily,VicENTE et al. (5) lave shown that urea, st>bstrate of t>rease, causesitself an aggregation of protein cooperating \vith tl>e L—usm,ic acid—

depending aggregatiom]. Tbe aggregates, commol]ly inactive, show a¡no!ect>lar wCi (r~>~ hí than

~ gher 800,000.TI>e imiactive aggregatesreverse to active protein when theaggre—

gation provoled by L—usnic acid is carried out in the presenceof 1-—

cysteim> (U). Ti>erefore. we shonld reject dic hypothesisreferring- to the

:íggregation state at tlie only responsiblefor the enzymatícinactivationsince active aggregatesof high molecular weight have been fonud.

The present paper studies tl,e changes in tl]e quaternary struciure

of t>rease produced by L—usnic acid depeuding on tIc tertíary structure

of el]zyme.

MATERIAl. ANT) METHOT)5

It lías beent>sed for tliis work crystalline urease(type II], Sigma Che-

mícal Co.) preinct>batedwith L—cystein and L—usmíic acid in the comícemí—tratiomí indicated in every case, as it has been previosly descrihed (2).

‘¡‘líe specific activity lías beendeterminedaccordingConway’smethod (7>estímating the quantity of protein accordimíg\Varburg a>,dCl>ristians (8)

60 1’ It-’ÉAJ LIS Diii. DPi.’ ErAMENTO DE EOr.XN 1 c,X ~ 1’ 1 510 LOt 1.’ X 1-Li; ¡VíA.. 001,. X

II]et I>o cl. TI me mnoleelijar WC ight of diffe rent aggccgates 1] ave beeu <leter—

>íiíued Iiy fi It ering tI] e samplci- tbrough a Seplía rose 613 coltunt> of 21cmi> i u Ile g it amuí 2 cm u diameter, XVI] ch xvas halancc ‘y itt 75 II> Nf

phosphatebííffer. pl—! 69, ¿md co>nparií>g eltíates ovitli a iltratiol> (ha—

grartí made umuler the saíneconditions eit her fo r t)-rOglob1>1 1>, gí tít afluí tedeIsvclroí~el/asi- - -.—t>rease and cacalase (alí fron, Signi;í Clic 1>3 cal C o -1-

40.0-

U]

ir *O

—MiO- 1

¡ £ 1

(3It

u 200

<~1o,o.U,

lo-o-

10 20 30 40 50 60 70 60

oc

- Figíire 1 —Tlíerrnic- iliactivaíioí] of arcase(II) híg iii 1(1 of

final ;-olííí>ie).

RESOL-rs ANt> DISCUSMItIN

1<) i>ig ti/-case tu a final of 10 >]1l are tl>em->]ticallv dc>t~ítti>-útl XVI>ití>ot>t

a precipitation of proceimí has beco carried oíít. Figure 1 sliows tl]cresuílts ohtaimíed froin tlíermic deactivation process aud figtíre 2 sliows

the resnlts of flltering tl]is protein umider different treatim]gs tl>rot>gl>Seplíarose<B - On it, it may be observeda continox>s appcaranceof twomalí> ínolect>lar forms, one of 820 000 molecular weight (eluítcd at 10

ml fi It cati011) amuí am,other, ti]e main pcale. of <190,000 niolectílar \Veigltt

ItrrECT Or L-Usxtc ACtO OK A TIIEÉMICALLY OENATURED U/IZASE 51

‘(eluted at 60 anl), Avhicla ever is the chemical treating tbe proteil] tl]er—

mically inactivated has suffered previonsly to filtratio>,. -

1-o

o,E

ir

o,oo.

1-o-

A a

Figure 2—Diagrain of liltration of a tl,errnicallvjílactivated torease: A) wl,ithout cl>emical trea—iÁíig B) incubated.br 5 mhattes at 370 C witl~L-usaic acitl (25 ~í moles/mí) C) with L-cys-1cm (50 ¡o onol es) and D) incubated x-i-iúí boíl>

L-usídc acid am>d L—cysteiu.

The enzyme is thcrmostable enough, since heing one bour at 70’ C

prox-okesthc loss of a third part of specific activíty. The total tl>ermic,

inactivation is achieved by incubation for oíoe hot>r at 80~90o E’.. Tuthis way, it is produced an aggregation stateof tI]ermic origín by whicli

-molecular weights similar to the ones obtained from tlie chemical mac—

¡‘y30 60 90 120150 30 60 90 120 150

filtrate 1 rn1

52 Ti? AV AJOS DEI, O FLCl TI ‘IV’ ‘>1 ICNTO Di? 1107ff LiL.X Y ‘15>OLO(Z./ A ~IZCETAT- - XO L - X

tivation processmay be obtained (5, 6). I>i ibis agrregatiol] state, >ieí—tl]er tl]e inactivator nor tbe amninoacid slsow considerableeffects s>nce

tíje peaks obtaíned by filtering tbroug-h SepharoseGB are identical to

the ones fou>]d for tl]e thermal denature(l e>]zyme xvben botí, com—

poc>ndsare absentfrom it. Finally, it coííld be deducedthat the tertiarystruct>ire >5 absolutelv í>ecessaryfor the i>]actix-ation process by L—c>s—

Ele acid to he carried ocft.

E? u’ =4 =4A 14 Y’

L—nsnic acid acts as an arcase inactívator cansng modifications o”tl]e quateri]ary strt>tcícti>re of the protein. E>ízyme tertiary strí>cture isabsolntely >]ecessary for the inactivation process-

REI’ErrNcIcs

(1) AIar,,l]ak, A. l7ager j - — 197,0 — j - CHI - Comp. Plívsiol 35.317.

(2; Viceí,te. (7. - Cucíva. U - ¿md Valle - Al. T. — ]073 — Rey. lEsma. Fisiol., 29. 29R.(3) Vicente. C., Guerra, H. and Valle Al. T. — 1974 — Rey. 1?síí. Fisiol. - 30. 1.(-4) Val] Sunícre. C. TV. AII,reclít, 1.. Declonder A. aad 14. (le if’ootcr aíid 1. Té — iii

Tbe CITÉI]:istrx- and llioclíemistr}- of Plant i’rotcins — (.1- E. Harbo¡i>e mdC. 1< Vaí> Sumere eds) — pp 211. Acaden,ic Press,New York.

(5) Vicente- C- , Azpiroz A, lfstevez, NI. E. at]d (lomízál ez - Al - L. — 11>78 — Plant -Ccli =íí,d llí, viroaíne,ít. 1 - 29.

(0) Y iccí/te C - nad Cifucí, tes E. — Pía,,t Soicuce T..ctters, iii lEe pren -(7) Coínv-ay-. II. 1. — 1 UflT — Aiicrodiffusión Analvs is aríd Volul],etric Erroí - Cros.

Ev Lookwood, 1.0 udoí>-

(8) \Varl,urg O raid Clíistiaíí. VV. — 11141 — Rocl>em. /, 384.