Phenylalanine ammonia-lyase entrapped in fibers

BIOCtlIMIE, 1980, 62, 575-580.

Phenylalanine ammonia-lyase entrapped in fibers. W. MARCONI, F. BARTOLI, R. GIANNA, F. MORISI and G. SPOTORNO.

Assoreni, Laboratories for biochemical Process, Monterotondo (Rome) Italy.

R~sumd.

Summary.

De la ph6nylalanine ammoniac-lyase fur immobilis6e sur des fibres de triac6tate de cellulose rendu h6mocompatible par m61anqe avec un antiaqr6qant plaquettaire.

Phenylalanine ammonia-lyase extracted from Rhodotorula rubra (IFO 1101) was immobilized into cellulose triacetate fibers made hemocompatible by physical blend with a platelet antia~qreqatinq aqent.

L'enzyme immobilis~e a pu aqir sur la ph& nylalanine et la tyrosine & leurs concentrations physioloqiques et n'en laisser que des traces apr~s quelques heures. pH optimum, rendement d'activit6 et stabilit6 ont 6t6 6tudi6s.

The entrapped enzyme could operate at physioloqical values of phenylalanine and tyrosine reducinq their level to traces within a few hours. The optimum pH value for the entrapped enzyme shifted from 8.0 to 9.0. At blood pH the activity was about 68 per cent of the maximum. The entrapped enzyme retained its oriqinal activity in blood for more than 50 days.

Introduction. P h e n y l a l a n i n e a m m o n i a - l y a s e (PAL) catalyzes the c o n v e r s i o n of p h e n y l a l a n i n e and t y r o s i n e to t r a n s - c i n n a m i c a c i d a n d t r a n s - c o u m a r i c acid, respectively. Some i n t e r e s t i n g a p p l i c a t i o n s of this enzyme can be e n v i s a g e d for the r e m o v a l of p h e n y l a l a n i ' n e a n d t y r o s i n e f r o m b l o o d o r food. Classic p h e n y l l c e t o n u r i a , t r a n s m i t t e d by an a u t o s o m a l r e c e s s i v e gene, results, from a defect in l i v e r p h e n y l a l a n i n e h y d r o x y l a s e [1]. This disease causes .an excess of p h e n y l a l a n i n e to accumulate in b l o o d and s p i n a l fluid. The disease develops significant i r r e v e r s i b l e b r a i n d a m a g e and behavioral derangement. To date, the only r e m e d y has been to p l a c e the i n d i v i d u a l s suffering from ~his disease on a diet l o w in p h e n y l a l a n i n e d u r i n g t h e i r first to t h i r d m o n t h of life [2]. F o r this r e a s o n a l n i n o a c i d m i x t u r e s d e p r i v e d of p h e n y l a l a n i n e are used as food, n o r m a l l y in con-

j u n c t i o n w i t h vitamins, m i n e r a l salts and small a m o u n t s of p r o t e i n s li~e casein. Some a u t h o r s have p o s t u l a t e d the potential a p p l i c a t i o n of PAL for the r e p r e s s i o n of some t u m o r a l cell growth. W. J. STITH et al. [3] s h o w e d that P A L effectively i n h i b i t s l e u k e m i c cells g r o w t h in vitro. M o r e o v e r h u m a n l y m p h o c y t e s grow on l)henylp y r u v a t e as a substitute for phenyl+alanine w h e r e a s l y m p h o b l a s t s do not. Consequently, by r e d u c t i o n of p h e n y I a l a n i n e p l a s m a levels t h r o u g h PAI. lreatm,ent and s u p p l e m e n t a t i o n w i t h p h e n y l p y r u v a t c , it m a y be possible to i n h i b i t n e o p l a s t i c ccI1 grovcth w i t h o u t a d v e r s e l y affecting the host. P A L t h e r a p y in vivo w a s also c o n s i d e r e d h i g h l y effective [4]. U n f o r t u n a t e l y P')~L is h i g h l y i m m u n o g e n i c w h e n inj,ected in r a b b i t s , and the o b t a i n e d a n t i b o d i e s p r e c i p i t a t e the enzyme. Then, r e p e a t e d i n j e c t i o n s of the enzyme b r o u g h t about a very r a p i d c l e a r a n c e of its a c l i v i t y in blood !5~. The t o x i c i t y of p u r i f i e d PAL is l o w e r than that observed w i t h partial,ly lmrified l)rel).arations but in lhis case also a mihl toxicity was observed. An immob i l i z e d enzyme might open i n t e r e s t i n g l)Crsl~eclives

576

E. Tiichsen and coll.

i n t h i s f i e l d b y o v e r c o m i n g s o m e p r o b l e m s s u c h as a n t i g e n i e i t y a n d e n z y m e t o x i c i t y e v e n if o t h e r p r o b l e m s a r e to b e s o l v e d s u c h as s u p p o r t t o x i c i t y and hemocompatibility. P A L w a s e n t r a p p e d in c e l l u l o s e t r i a c e t a t e f i b e r s b y t h e p r o c e s s d e v e l o p e d ,at A s s o r e n i ' s L a b o r a t o r i e s . T h e p r o p e r t i e s of , e n t r a p p e d e n z y m e ~vere i n v e s t i g a t e d in c o m p a r i s o n w i t h t h e f r e e e n z y m e . A c t i v i t y t e s t s w e r e d o n e in vitro i n b l o o d w i t h fibers m a d e h e m o c o m p a t i b l e by a p r e v i o u s l y desc r i b e d m e t h o d [6]. T h e r e s u l t s d e m o n s t r a t e d t h e f e a s i b i l i t y o f t h i s t r e a t m e n t f o r a n in v i o o a p p l i cation.

Materials and Methods. Materials. L-phenylalanine, L-tyrosine, t r a n s - c i n n a mic acid, t r a n s - c o u m a r i c acid and cellulose triacetate (CTA) were purchased f r o m Fluka (Fluka AG Buchs Switzerland). P r o t a m i n e sulfate and t r i s o d i u m citrate were purchased f r o m Merck (Merck D a r m s t a d t West Germany). Methylene chloride, toluene and o t h e r chemicals were reagent grade f r o m C. Erba, Milan, Italy. The 4-5 diphenyl-2~bis (2-hydroxyethyl) aminooxazole (ditazol) used for the p r e p a r a t i o n of t h r o m b o r e s i s t a n t fibers was prepared according to a procedure described in the literature [7].

literature [8]. 650 g of wet cells were suspended in 2500 ml of 50 mM t r i s buffer, pH 8.5 and disrupted by a Gaulin h o m o g e n i z e r M15 (Gaulin Corporation, Everett, Massachusetts, USA). The suspension was then centrifuged at 16.000 × g for 20 rain using a Sorvall centrifuge (RC2B). The precipitate was w a s h e d w i t h 100 ml of tris buffer a n d centrifuged again. To the pooled s u p e r n a t a n t s (310{) ml), 200 ml of p r o t a m i n sulfate solution (5 per cent) a d j u s t e d to pH 8.5 with a m m o n i u m hydroxide, were added u n d e r stirring. The ratio p r o t a m i n e sulfate to protein was 155 rag/ gr. The m i x t u r e was centrifuged at 2.500 × g for 20 min. To the s u p e r n a t a n t 1550g of a m m o n i u m sulfate (78 per cent saturation) were added. After centrifugation (2600 × g for 20 min), the recovered precipitate was dissolved in 500 ml of tris buffer, treated with 110 g of a m m o n i u m sulfate (39 per cent saturation) and centrifuged (2500 × g). This second precipitate was dissolved in 165 ml of tris buffer and dialyzed against 5 liters of the same buffer. The dialyzed solution (210 ml) was stepwise fraetionated b y adding t r i s o d i u m citrate. The active fractions were p r e c i p i t a t e d w h e n the a m o u n t of sodium citrate was in the range of 17.5 - - 33.5 g/ 1{)0 ml. The active fractions, recovered by cen,trifugation (25000 × g) were dissolved in t r i s buffer and dialyzed against the same buffer. For the e n t r a p m e n t and kinetic evaluations, the fraction precipitated in the range 2~1.5-25 g/100 ml citrate was used. All t h e purification steps were done at 4°C. The results obtain,ed in a typical purification procedure are reported in table I. Enzyme assay. - - The activity of free and entrapped enzyme on p h e n y l a l a n i n e was assayed [9] by measu-

TABLE I.

P h e n g l a l a n i n e a m m o n i a tgase p u r i f i c a t i o n . Protein (my)

Activity (U}

Sonic extract

62,930

7,690

0.1

--

Protamine sulfate (supernatant)

28,150

7,615

0.27

99

Ammonium sulfate precipitate (concentration step)

21,640

7,580

0.35

99

Ammonium sulfate (39 per cent of saturation) (precipitate)

14,465

6,365

0.44

83

1,881

3,480

1.85

45

Purification step

Sodium citrate (21.5 - 25 g/100 ml) (precipitate)

Enzymes. Rhodotorula rubra (IFO 1101) was m a i n tained on 3 per cent agar slants containing 1 per cent malt extract and 0.1 per cent yeast extract. The yeast was cultured on a m e d i u m of 1 per cent Difeo yeast extract, and 0.1 per cent DL-phenylalanine in tap w a t e r [8]. The extraction and purification of PAl. were similar to other procedures reported in the BIOCHIMIE, 1980, 62, n ° 8-9.

Specific activity (U/mg protein)

Yield

per cent

ring the f o r m a t i o n of t r a n s - c i n n a m i c acid monitored by increasing the absorbance at 29O nm (Pye Unicam SP 1800). The molar extinction coefficient (e) of cinnamic acid at 290 n m is 10.000 (liter M 1 cm-D. The same was done using t y r o s i n e as substrate and measuring the absorbance of t r a n s - e o u m a r i e acid at 315 n m (e ---- 7.400 liter M-1 era-i).

Phentllalanine ammonia-lyase The reaction mixture contained 0.833 mM phenylalanine in 0.05 M tris buffer, pH 8.5. The reaction began by addition of the enzyme to the substrate solution at 30°C. One enzyme unit is defined as the amount of protein which catalyzes the formation of I lamole of cinnamate per rain a t 36°C. The specifie activity is expressed as the i~moles of einnamate formed per min at 30°C by 1 mg of proteiu or, in the case of entrapped enzyme, by 1 g of dry material. The activity determinations in blood were carried out by determining the amount of non transformed aminoaeid. After centrifugation of citrated blood at 3000 rpm for 10 rain, the recovered plasma was deproteinized with 50 mg of sulfosalicylie acid per mr. The aminoacids in the supernatant were determined by a C. Erba rood. 3A27 aminoacid-autoanalyzer. Proteins were determined by the Lowry method [10].

Results.

PREPARATION OF THE FIBERS. T h e fiber e n t r a p p e d P A L w a s p r e p a r e d by a s i m p l e m o d i f i c a t i o n of the p r e v i o u s l y d e s c r i b e d g e n e r a l m e t h o d [11]. T h e m o d i f i c a t i o n c o n s i s t e d of a d d i n g to the p o l y m e r p h a s e , b e f o r e s p i n n i n g , t h e p l a t e l e t antia g g r e g a t i n g a g e n t (10 p e r c e n t w i t h r e s p e c t to the cellulose triacetate).

entrapped

in f i b e r s .

577

4~ o

2C

I

I

pH

Fro. 1. - - Effect of the reaction mixture pH on PAl, activity. The activity of free enzyme ( - - O - - ) was measured at 37°C in 3 ml of 0.025 M tris and 0.025 M phosphate buffer containing 50 mM phenylalanine. The amount of enzyme used for testing was 2() ~g. For entrapped PAL (@--@), 50 mg of fibers containing 220 t~g of protein were used. The reaction was performed at 37°C under stirring. The absorbance changes over a time period of 10 min were measured, at 1 minute intervals, by sampling 3 ml of the reaction mixture.

4

3

T h i s w a s d o n e b e c a u s e in vivo tests s h o w e d that t h e p h y s i c a l b l e n d of t h e c e l l u l o s e a c e t a t e and p l a t e l e t a g g r e g a t i n g agents gave h e m o c o m p a tible fibers [6, 12]. T h e a m o u n t of e n t r a p p e d e n z y m e w a s 8.8 m g p r o t e i n / g C T A c o r r e s p o n d i n g to 24.'6 U / g CTA. __

EFFECT OF in t h e r a n g e b u f f e r (tris 0.025 M) a n d

pH. T h e p H effect w a s t e s t e d at 37°C of 5 - 11 w i t h 0.05 M i r i s - p h o s p h a t e 0.025 M + p o t a s s i u m p h o s p h a t e 50 m M L - p h e n y l a l a n i n e .

T h e results o b t a i n e d a r e r e p o r t e d in figure 1. T h e f r e e e n z y m e s h o w s a p H o p t i m u m at 8.0. T h e o p t i m u m p H for t h e e n t r a p p e d e n z y m e is s h i f t e d to 9.0. TEMPERATURE EFFECT. T h e effect of t e m p e r a t u r e on t h e e n z y m e a c t i v i t y w a s t e s t e d w i t h 50 mM p h e n y l a l a n i n e in 0.05 M tris, p H 8.5 in the r a n g e of 10 - 4 0 ° C . F r o m the Mope of t h e A r r h e n i u s plots, figure 2 1

(In v vs~-KK), a c t i v a t i o n

energies

of 12,0'54 and

12,840 c a l / M o l e f o r the f r e e a n d e n t r a p p e d P A L were respectively calculated. T h e t e m p e r a t u r e e f f e c t on t h e e n z y m e s t a b i l i t y w a s also tested.

BIOCHIMIE,

1980, 62, n ° 8-9.

i 315

i

i 325

i

i 33,5

i

i 345

i

i 355

-I.10

~

T

Fro. 2. - - Arrhenius plots for free ( 0 - - 0 ) and entrapped PAL (O--O).

S a m p l e s of f r e e a n d e n t r a p p e d e n z y m e w e r e s t o r e d f o r 30 m i n at d i f f e r e n t t e m p e r a t u r e s in 0.0~5 M tris b u f f e r p H 7:5 a n d 8.5. T h e r e s i d u a l a c t i v i t y w a s d e t e r m i n e d at 37°C w i t h 50 mM L - p h e n y l a l a n i n ' e in 0.05 M l r i s - H C l , p H 8.'5. T h e r e s u l t s are r e p o r t e d in figure 3. SUBSTRATE CONCENTRATION. T h e a c t i v i t y of f r e e a n d e n t r a p p e d PA'L w a s t e s t e d at d i f f e r e n t c o n c e n t r a t i o n s of L - p h e n y l a l a n i n e a n d L-tyrosine in 0.0*5 M t r i s - H C l buffer, p H 8.5, at 37°C. U s i n g t y r o s i n e as s u b s t r a t e in t h e r a n g e 5 " 1.000 ~M, l i n e a r d o u b l e r e c i p r o c a l plots w e r e o b t a i n e d b o t h for f r e e a n d e n t r a p p e d P A L (figure 4).

578

W . Marconi a n d coll. The double reciprocal plot for free PAL using phenylalanine as substrate in-the range 0,00,5 . ' 1 0 0 raM is s h o w n i n f i g u r e 5. F i g u r e 6 shows the relationship between reaction rate and phenylalanine concentration for entrapped PAL.

~oq.

sq. ¢ o

~

F

2.0_

T (°C)

FIG. 3. - - T h e r m o s t a b i l i t y o f free a n d e n t r a p p e d p h e n y l a l a n i n e anarnonia lyase at pH 7.5 and 8.5. The e n z y m e p r e p a r a t i o n s were h e a t e d a t the indicated t e m p e r a t u r e f o r 30 m i n u t e s in 0.0.5 M tris buffer, pH 7.5 a n d 8.5. The r e s i d u a l activity of the e n z y m e were assayed at 37°C w i t h 50 mM p h e n y l a l a n i n e in 0.05 M tris buffer pH 8.5. The symbols (O--O) a n d (O--D) indicate the results for free enzyme at pH 7.5 a n d 8.5, respectively ; the symbols (@--@) a n d (&--&) indicate t h e results for e n t r a p p e d PAL at pH 7.5 a n d 8.5, respectively.

30

V

0,5~

1

2

3

4

substrate

5

6

7

S

30

70

110

concentrationlmM}

Fro. 6. - - A c t i v i t y of f i b e r entrapped PAL vs the phen y l a l a n i n e concentration. The m e a s u r e m e n t s were done at 37°C in stirred batches using 50 mg of fiber in 50 ml tris buffer pH 8.5. The a b s o r b a n c e change over a t i m e period of ]0 rain, at 1 rain intervals, were m e a s u r e d by s a m p l i n g 3 m l of the reaction m i x t u r e . The symbol (&) refers to tests w i t h fibres containing ditazol. The symbol (@) refers to tests w i t h fibers made w i t h o u t ditazol.

20

40

10

2500

5 O0

1_

7500

S

FIG. 4. - - L i n e a w e a v e r - B u r k double reciprocal plot f o r free (&--&) and entrapped ( ~ - - D ) P A L using tyrosine as substrate.

Flo. 7. - - L i n e a w e a v e r - B u r k plot f o r free PAL, obtained using phenlllalanine as substrate w i t h o u t cinnamic acid (O----O), w i t h 5,0 ~M (&--&) a n d w i t h 100 ,~M ( ~ - - D ) of e i n n a m i c acid, respectively.

3

7 2

1

5000

10000

15000

"1 S

Fro. 5. - - L i n e a w e a v e r - B u r k double reciprocal plot for free P A L w i t h p h e n y l a l a n i n e . BIOCHIMIE, 1980, 62, n ° 8-9.

I n t h i s c a s e t h e r e c i p r o c a l p l o t ( 1 / v vs l / S ) gave no linear relationship. It was ascribed to the c o m p e t i t i v e inhibi,t,ion of ~ i n n a m i c a c i d p r o d u c e d i n t h e f i b e r m i c r o c a v i t i e s w h o s e e f f e c t is s t r o n g e r at l o w s u b s t r a t e c o n c e n t r a t i o n s . F o r t h i s p r o d u c t a Ki of 19 ~M w a s c a l c u l a t e d {fig. 7) w i t h t h e f r e c P A L . T h e k i n e t i c s val.ues c a l c u l a t e d f r o m t h e p l o t s a r e r e p o r t e d i n t a b l e H.

P h e n y l a l a n i n e a m m o n i a - l y a s e e n t r a p p e d in fibers.

579

T~mLE II.

Kinetic parameters for free and entrapped PAL. Free PAL

Vm (~moles/min X m g protein)

Substrate

Entrapped PAL Km (~M)

Tyrosine

0.24

167

Phenylalanine

2.83

439

Ki (.~M)

Vm (umoles/mio × m g protein)

Km (~M)

0.13

370

2.62

Cinnamic acid

19

STABILITY OF ENTRAPPED AND FREE PAL. F o r the i m m o b i l i z e d e n z y m e s it is of o v e r w h e l m i n g imp o r t a n c e to k n o w the a c t i v i t y c o n s t a n c e both u n d e r w o r k i n g and trader storage c o n d i t i o n s . F r e e and e n t r a p p e d PAL s~amples w e r e stored at 4°C in closed bottles. T h e r e s i d u a l e n z y m e a c t i v i t y w a s m e a s u r e d d~aring a p e r i o d of two years. F i g u r e 8 s h o w s the residual activities vs the storage time.

PAL e n t r a p p e d in fibers was tested in buffer solution at pH 7:5, 8.'5 and in blood. F o r the test in buffer solution, two samples of 3.1 g of d r y fibers w e r e p a c k e d in t w o j a c k e t e d ccdumus ( d i a m e t e r 13 mm, l e n g t h 145 ram). T h e substrate solutions c o n t a i n i n g 10 mM L-phenylalanine in 0.05 M tris buffer pH 7.5 and pH 8.6 w e r e fed at 37°C and at a flow rate of 1 m l / m i n . In the outlet the c o n v e r sion of the substrate w a s m e a s u r e d . T h e p e r c e n t of residual r e p o r t e d in figure 9.

a c t i v i t y vs time

is

T h e stability test in blood was done in a s t i r r e d b a t c h at 37°C w i t h 200 mg of d r y fibers in 25 ml of c i t r a t e d blood, w h o s e c o n t e n t of p h e n y l a l a n i n e and t y r o s i n e was i n c r e a s e d till 0.,5 mM.

v

so

i 0 5

i

i

i

1.0

1.5

2D

E v e r y 24 h o u r s the b l o o d w a s substituted w i t h fresh blood.

r e m o v e d and

T i m e (years)

Fro. 8. - - Stability of free (©) and entrapped PAL (0) us storage time. 80

>

60

~w 4o 20

10

2

0

40

50

Time (days)

1'o

2b

3~o

40

-5'0

Time (days)

FIG. 9. - - Stability of entrapped PAL under working conditions in buffer, at pH 7.5 ( 0 - - 0 ) and 8.5 (m--B) using phenylalanine as substrate.

STABILITY

OF ENTRAPPED

PAL

IN BUFFER

AND

IN

BLOOD. The stability u n d e r w o r k i n g c o n d i t i o n s of

BIOCHIMIE, 1980, 62, n ° 8-9.

Fro. 10. - - Stability of entrapped PAL under working conditions in blood. The values of conversion for phenylalanine (A) and tyrosine (O) after 60 min of reaction for each batch vs the working days are reported.

C o n v e r s i o n d e t e r m i n a t i o n s w e r e done the first 120 m i n for each batch.

during 39

580

W . M a r c o n i a n d coll.

The residual activity vs the time is r e p o r t e d i n figure 10 and the typical kinetic curves o b t a i n e d in citrated blood w i t h ,entrapped PAL are reported i n figure 11.

F o r b i o m e d i c a l applications, the toxicity not only of i m p l a n t a b l e p o l y m e r s but also of monomers and additives that can be released is an i m p o r t a n t problem. The p o l y m e r used for the e n t r a p m e n t , cellulose triacetate, is safe. No addifives such as plasticizers are requested for the fibers.

1OO_

a

o

_

~

60_

20_

a'o

e~

9h

~to

T i m e (m~r~

Fro. 11. - - Typical kinetic carves for phenglalanine (&) and tgrosine (@) obtained in blood with entrapped PAL.

Discussion.

PAL from R h o d o t o r u l a r u b r a extracted and purified w i t h a simple p r o c e d u r e was successfully e n t r a p p e d in cellulose triacetate fibers. The specific a c t i v i t y was 1.8'5 U / m g protein. F r o m L i n e w e a v e r - B u r k double r e c i p r o c a l plots, a Vmax of 2.83 and 0.24 U/rag p r o t e i n was calculated for p h e n y l a l a n i n e and tyrosine, respectiv.ely. The free enzyme ~vas quite slable u n d e r storage c o n d i t i o n s a n d this~ stability was e n h a n c e d by the entrapment. The e n t r a p p e d enzyme showed a small shift of o p t i m u m pH from 8.0 to 9.0. This m i g h t be attributed to the local higher c o n c e n t r a t i o n s of c i n n a mic acid inside the m i c r o c a v i t i e s w h e r e the enzyme is located c a u s i n g a pH gradient. At physiologic,al values (pH 7..5) the activity is. about 68 per cent of the max.imum, a r e a s o n a b l y high activity for practical applications. C i n n a m i e acid is also a competitive i n h i b i t o r of PAL (table II) and, due to diffusional limitations, the a c c u m u l a t i o n of c i n n a m i e acid inside the mLcrocavities of ihe fibers, could be respon~sible for the shift of the m a x i m u m activity towards very high values of s,ubstrate concentrations.

BIOCHIMIE,

1980, 62, n ° 8-9.

U n f o r t u n a t e l y this p o l y m e r gives rise to thrombus f o r m a t i o n in blood. However in v i v o tests in blood showed that the p h y s i c a l blend of the polymer w i t h an antiaggregating agent such as ditazol completely suppressed the t h r o m b u s formation. Moreover the b l e n d w i t h the ditazol did n.ot affect the fibers efficiency, as s h o w n in figure 6. The enzyme is not in d:irect co.ntact with thee e x t e r n a l fluid, but o n l y nvith low m o l e c u l a r weight substances that can enter the i n t e r n a l m i c r o c a v i t i e s of the fibers, from w h i c h the enzyme c a n n o t escape because of its relatively big size. F o r this reason, this i m m o b i l i z a t i o n system p r e v e n t s i m m u nological reactions a n d toxic effects of the enzyme preparation. In conclus,ion, this study demonstrates that PAL e n t r a p p e d i n h e m o c o m p a t i b l e cellulose acetate fibers could be a n e w tool in the t r e a t m e n t of p h e n y l k e t o n u r i a disease or in the p r e v e n t i o n of leul~emic cell growth. F u r t h e r studies in a n i m a l s are r e q u i r e d to determ i n e w h e t h e r this i m m o b i l i z e d system might or not be a useful agent for the treatment in man. REFERENCES. 1. Udenflend, S. a Bessman, S. P. (1953) J. Biol. Chem., 203, 961. 2. Hsia, D. Y. ~ Holtzmann (19.73) in <~Medical Geneties >>, Me Kusick V. A. a Claiborne R. Eds. (HP Publisking N.Y.) 237-244. 3. Stith W. J., Hodgins, D. S. ~ Abell, C. W. (1973) Cancer Res., 33, 966-971. 4. Abell, C. W., Hodgins, D. S. ~ Stith, W. J. (1973) Cancer Res., 33, 2529-2532. 5. Fritz, R. R., Hodgins, D. S. ~ Abell, C. W. (1976) J. Biol. Chem., 251, 464~6-4650. 6. Bartoli, F., Giovenco, S., Lostia, 0., Marconi, W., Morisi, F., Pittalis, F., Prosperi, G. ~ Spotorno, G. (1977) Pharm. Res. Commun., 9, N. 6, 521-546. 7. Marehetti, E., Mattalia, G. ~ Rosati, V. (1968) J. Med. Chem., 11, 10.92-1093. 8. Hodgins, D. S. (1971) J. Biol. Chem., 246, 2977-2985. 9. Hodgins, D. S. (1968) Biochem. Biophys. Res. Commun., 32, 246-253. 10. Lowry, O. H., F arr, A. L., Randall, R. J. ~ Rosebrough, N. J. (1951) J. Biol. Chem., 193, 265-275. 11. Dinelli, D. (1972) Proc. Biochem., 7, N. 8, 9-12. 12. Marconi, W. E n z y m e Engineering, Broun, Maneeke Wingard eds. Vol. 4, 179-186.