Effects of surface active agents on crystalline rabbit muscle phosphorylase

554

I~IOCHIMICA El" BIOPHYSI(A AC'I'A

VOI,.

17 (.955)

E F F E C T S O F S U R F A C E A C T I V E A G E N T S ON C R Y S T A L L I N E R A B B I T MUSCLE P H O S P H O R Y L A S E l)v R O B E R T \V. COXVGILI~*

Department o~ t3iological Chemislry, Washinglo~t Universilv School o~ Medicine, .';l. Louis, 31issoltri (I:.S..1.)

Many examples of denaturation of enzymes by detergents or surface active agents (SAA) are known (PuTNA~I14). Few instances of activation of enzymes by such agents have been reported. ALLEN AND ItlODINE 1 w e r e able to activate protyrosinase of grasshopper eggs by anionic alkyl sulfates. Cationic alkyl amines were ineffective or inhibitory. On the other hand, KREBSH and HIrC;HES'~reported that certain cationic SAA increased the activities of bacterial glutamic acid decarboxylase and glutaminase while anionic SAA inhibited these enzymes. Nonionic SAA were without effect in the few cases where tests have been reported. Cationic, anionic and nonionic SAA in dilute solution display anomalies in certain physicochemical properties, such as viscosity, surface tension, equiwdent conductivity, freezing point lowering, etc., with change of concentration. These anomalies are interpreted as arising from the association of molecules of the SAA into aggregates or micelles, McB~\INx2. This transition is rather abrupt with change of concentration of the SAA and the concentration at which the anomalies appear is referred to as the critical micellc formation concentration (CMC). These agents exert their most profound effects upon proteins when present in such amounts that micelles exist. Indeed, HUt;HES1° found that the minimum concentrations of cationic SAA required to activate glutamic acid decarboxylase and glutaminase coincide with their CMC wdues. M:\THEWSla has shown a correlation between the CMC values of several anionic SAA and the concentrations of these SAA for equivalent inhil)ition of hwdnronidase. Crystalline rabbit muscle phosphorylase is activated by nonionic surface active agents. Cationic and anionic SAA inhibit this enzyme. The importance of the micellar structure of the SAA for actiwition or inhibition is shown by the facts that the concentrations of nonionic SAA for activation and of cationic SAA for inhibition coincide with their concentrations for micellc formation. Further, certain nonionic SAA which do not form micelles fail to activate rabbit muscle t)host)horylase. MATERIALS .\NI) METHODS Surface active agents were obtained from the following sources. Atlas I'owder C o m p a n y supplied the nonionic agents Tween 8o (polyoxyethylene sorbitan monooleate), Myrj 51 (polyoxyethylene stearate), Brij 35 (polyoxyethylene lauryl alcohol), Span 2o (sorbitan monolaurate), .\rlacel C * This research was completed in ~952 during the tenure of a Public Health Service l~ostdoctorate Research l:ellowship from the Xational Cancer Institute. I ' r e s e n t address, D e p a r t m e n t of Bi~chemistry, L!niversity of California, Berkeley 4, California.

Re/erences p. 560.

v o L . 17

(1955)

SURFACE ACTIVE AGENTS AND RABBIT MUSCLE PHOSPHORYLASE

555

(sorbitan sesquioleate), G - I I 4 4 (polyoxyethylene sorbitol oleate), and G-224o (polyoxyethylene sorbitol). Polyethylene glycols (PEG) of various average molecular weights were furnished b y Dow Chemical Company. The average molecular weight as reported b y the supplier is designated by the sample n u m b e r , i.e., PEG-4oo is a sample of av. nlol. w t 4oo. Triton X - I o o is a nonionic alkyl aryl polyethylene glycol supplied b y R o h m and H a a s C o m p a n y . Union Carbide and Carbon Corporation furnished the cationic agent, Amine 22o. The anionic agent Aerosol O.T. (dioctyl sodiunl sulfosuccinate) was purchased from E i m e r and A m e n d C o m p a n y and the cationic agent, cetyldimethyle t h y l a n l m o n i u m b r o m i d e (CDEA), from E a s t m a n K o d a k C o m p a n y . D e t e r m i n a t i o n s of critical micelle-fornlation concentration (CMC) were made by a modification of the nlethod of CORRIN AND HARKINS4. This m e t h o d is based on the color change t h a t an a p p r o p r i a t e acid-base indicator will undergo, even in a well-buffered solution, when a SAA is added in a n l o u n t s equal to or in excess of the CMC. A solution of o.ool 5 o,{~ by weight b r o m o t h y m o l b l u e (Eimer and A m e n d Company) in o.o 3 M imidazole buffer of p H 6.8 was used in the present experiments. A m a r k e d color change from the greenish-blue t h a t is characteristic of b r o m o t h y m o l b l u e at p H 6.8 to a b r i g h t yellow occurred w h e n a nonionic or anionic SAA (previously adjusted to p H (i.8 when tested with a glass electrode p H nleter) was added above the CMC. Cationic SAA such as CDEA or Amine 22o altered the color to a brilliant blue. A series of samples of c o n s t a n t composition, excep for progressive dilution of the SAA, were c o m p a r e d in a K l e t t - S u m m e r s o n photoelectric colorimete~ (Klett filter No. 56). The c o n c e n t r a t i o n of SAA required for half the m a x i n l u m color transfornlation was selected as the CMC. R a b b i t muscle p h o s p h o r y l a s e was crystallized by the m e t h o d of GREEN AND CORI8. P h o s p h o r y lase activity is expressed in the units defined by CoRI, CORI AND (~REEN2. N u m h e r of units = 1ooo ;~ K , where, K = I / l log

(1)

x~ Xe

X

The p e r c e n t g l u c o s e - l - p h o s p h a t e (G-I-POa) converted to polysaccharide in time, l, is represented by x, while xr is the percent converted to polysaccharide at equilibrium. Glycogen was present in excess. The m e a s u r e of activity was the rate of inorganic p h o s p h a t e liberation during the reaction. The p h o s p h o r y l a s e p r e p a r a t i o n was p r e i n c u b a t e d at 30 ° C with O.Ol 5 M eysteine at p H 6.8 for fifteen minutes. The p r e i n c u b a t e d enzyme was added to a solution of g l u c o s e - l - p h o s p h a t e and glycogen in such a m o u n t s t h a t the u l t i m a t e conlposition of the assay m i x t u r e was enzyme, 0.0075 Al cysteine, O.Ol 5 A1 G - I - P O 4 and i O/~oglycogen at p H 6.8. I n c u b a t i o n was continued at 3°`' and samples were w i t h d r a w n at a p p r o p r i a t e intervals for d e t e r m i n a t i o n of inorganic p h o s p h a t e b y the m e t h o d of FISKE AND SUBBAROW6. F o r d e t e r m i n a t i o n of total p h o s p h o r y l a s e (phosphorylase a + pllosphorylase b), the assay m i x t u r e also contained i x IO 3 2}I muscle adenylic acid (AMP). P h o s p h o r y l a s e a / p h o s p h o r y l a s e (a F b) corresponds to the ratio of activities found in the absence and in the presence of AMP respectively. RESULTS

Crystalline rabbit muscle phosphorylase could be activated as much as two-fold by the additon of certain nonionic SAA such as Tween 8o during the preincubation period with cysteine. The data of Table I indicate that the activation is a relatively slow process. Cationic and anionic SAA inhibit the enzyme strongly. Activation of both phosphorylase a and phosphorylase b by a nonionic SAA could be demonstrated by the experiment of Fig. I. Phosphorylase a is converted to phosphorylase b by the PR enzyme isolated from rabbit muscle a. Prior to exposure of crystalline phosphorylase a to PR enzyme, Tween 8o activated the enzyme 19o % in the absence of AMP and 15O°,o with AMP present in the assay mixture. After incubation of the phosphorylase with PR for varying periods, aliquots of the mixture had progressively diminishing activity in the absence of AMP (conw?rsion of phosphorylase a to phosphorylase b) but the per cent activation by Tween 8o in the presence of AMP in the assay mixture remained at about 15o%. It is unlikely that the nonionic SAA merely stabilizes the enzyme and thereby prevents denaturation during the preincubation and assay periods. Phosphorylase decreases in activity only lO% during as long as three hours preincubation at 3 0o and the same loss occurred in the presence of Tween 8o. Furthermore, the percent activation by Re/erences p. 560.

556

R.w.

COW(;U~L

T,\ HI.E

V()L. 17 ( I 9 5 5 )

[

EIrFI~2CT OF TIME OF INCUBATION \VITtI "I'\VEIg-N ~O ON THF ]~2XTENT OF ACTIVATION OF CRYSTALLINE RABBIT PHOSPHORYLASE

All enzyme samples were preincubated with o.ot 5 .ll cysteine at 3°` for 15 m i n u t e s prior to addition to an equal vohlme of the (;-I-P()1 and glycogen mixture.

I'k~sphoryh*.sca

('lmdilions

A SS(tl' qtOn])l~' [l'~"

gtC/lI'[lU

I :~11'[¢

I~gfl?etZ[ ctCli;'llV

5 minutes i o llli n u t e s

I 2.2 13 "4

i oo

J5 m i n u t e s

i4. 4

o.o[ ('0 Tween 8o adde(1 to the ( ; - I - l ' ( ) l and glycogen m i x t u r e t rain before the end of preincubation

5 minutes io minut:es i .5 minutes

[4.8 18.i IS. 9

o . o [ % Tween So a(lded to the (; I-I'O 4 and glycogen m i x t u r e at the s t a r t (7t" 15 rain preincubati(m

5 minutes Io minutes

1().8

I5 minutes

-(7. 7

o . o I o,, T w e e n S o ad(le(1 t(7 t h e e n z y m e a n d ( y s t e i n e m i x t u r e 5 r a i n b e f o r e t h e e n d (Tf t h e p r e m c u b a t i o n

5 minutes ]o m i n u t e s t 5 minutes

"°.7 co. 5

o.ol o,,o Tween 8o a(lded to the enzyme_ and cysteine m i x t u r e at the s t a r t of 15 rain preincul)ation

5 milmtes ]o milmtes 15 minutes

2 1 ,O 2 1 .(7 2I.I

N o Tween 8o added

I3O

I S.9

I 0.

I,to

(7 J.55

100

[ O~

50

"~ ~ "~0... ["

-o ......

o

F

O

c~

O

Fig. [. Action of I)R enzyme on phosphorylase a: Effect on p h o s p h o r y l a s e activation by Tween 8o. Salnples were assayed for phosphorylase activity after 5' incubation at 3° with o.o 3 31 cysteine and o.] 31 Nal: :[ 0.02% Tween 80 and ] I' I(7 a ,11 AMP.

30 "5

,

O._

ao LI.N.

'e

~'0

D

I0

"'"-

"'~*~" "---e~:

"I

~:

t

\"

0

io 20 30 40 Time of Incubation ot 3 0 ° C with PR

I

Tu'cen ,,,%

A MI )

1

P

.... • I

-

50 ......

t

(Mins.)

T w e e n 80 w a s t h e s a m e w h e t h e r i n c u b a t i o n a n d a s s a y w a s a : 8 ° o r 30% a n d w a s c o n s t a n t over a seven-fold change in phosphorylase

concentration.

A l s o e x c l u d e d is a n i n f l u e n c e

of t h e S A A o n t h e r e l a t i o n of e n z y m i c a c t i v i t y t o t h e p r e s e n c e o r a b s e n c e of a d e n y l i c acid, inosinic acid or cysteine; glycogen concentration (o.oo5-o.o53i);

l?e/eremes p. 560.

o r p H of t h e r e a c t i o n m i x t u r e

(o-2.5'?,;)" G - I - P

Activation

concentration

also seemed independent

of

VOL. 17 (i955) SURFACE ACTIVE AGENTS AND RABBIT MUSCLE PHOSPHORYLASE

557

the condition of the phosphorylase crystals such as n u m b e r of recrystallizations, age of the p r e p a r a t i o n or the ratio of phosphorylase a/phosphorylase (a _L b). Not all of the nonionic SAA increase the a c t i v i t y of crystalline r a b b i t muscle phosphorylase although the change in surface t e n s i o n m a y be of the same order. For example, Tween 80 a n d polyethylene glycol, PE(;-4oo , both lower the surface t e n s i o n of aqueous solutions to 35-4 ° dynes/cm. Yet PE(i-4oo had no influence on phosphorylase a c t i v i t y even when present in a m o u n t s up to 1%. The higher molecular weight PE(;-4ooo , however, produced a c t i v a t i o n a b o u t e q u i v a l e n t to t h a t b y Tween 8o (Table II) as t h o u g h the TABI.E If COMPARISON

OF T H E

ACTION

OF S U R F A C E AND

Activily o/ Agent

phosphorylast*

None Tween 80 Myrj 51 Brij 35 Triton Xqoo

100 ~{, 18o i8o 16o 155

G-1144 Span 2o Arlacel C PEG--, oo PEG-4oo PEG-6oo PEG-4ooo

145 15° t4o ioo i oo i ~5 I5o

THE

ACTIVE AGENTS

A B I L I T Y TO

FORM

ON

RABBIT MUSCLE

A orion ¢,n t romothymol6lue at p H 6.,~ Color clt *n',..'e

Deep Deep Deep Deep

yelh~w yellow yellow yellow

CMC

o.ooS o~ 0.006°o o.oi 4 (~ o.o t 7 °o

(;-2240

Aerosol O.T. Amine z2o CDEA

Puhtished values CMC:

Re/crence

(wt) (wt) (wt) (wt)

(3" t o ~ M )

l)eep yellow Slight yellow* * Slight yelh)w** No change No change No change No change 85 No change Strong inhibition Slight yellow Inhibition Deep blue Inhibition Deep blue

PHOSPttORYLASE

MICELLES

9" IO- 4 31

v

o.ot 3I

l~

o.o I t o~ (wt)

o.oo8:ll 8. l o s 31 I • IO a 3I

* Enzyme incubated with SAA plus cysteine for 15 minutes. Cone. of SA.\ was 0.04 o~,; except for PEG-4oo (I o.'), and Aerosol, Amine 22o and CDEA (o.oo02 M). ** Span 2o and Arlacel C were too insoluble for CMC determination. greater size of the polymer were i m p o r t a n t . This possibility is of interest in light of the observation of HU(;HES 1° t h a t the m i n i m u m c o n c e n t r a t i o n of cationic SAA for a c t i v a t i o n of bacterial glutamic acid decarboxylase a n d g l u t a m i n a s e coincides with the concentration required for creation of micellar structures within the solution b y aggregation of the SAA. It m a y be seen from Table II t h a t those SAA which activated phosphorylase are in all cases, except for the higher molecular weight polyethylene glycols, the same agents t h a t gave evidence of micelle-formation b y change in color of b r o m o t h y m o l b l u e . Tile d a t a for d e t e r m i n a t i o n of the CMC of Tween 8o a n d superposition of d a t a for a c t i v a t i o n of r a b b i t muscle phosphorylase b y this agent are given in Fig. 2. T h a t the c o n c e n t r a t i o n of the cationic agent, CDEA, for i n h i b i t i o n of r a b b i t muscle phosphorylase also coincides with its CMC is indicated b y Fig. 3. Crystalline r a b b i t muscle phosphorylase was recrystallized from a solution of o.o73/ cysteine a n d o.o3 M//-glycerol phosphate, p H 6.8, to which was added Tween 8o to o.o2 % (an a m o u n t in excess of the CMC). The recrystallized p r e p a r a t i o n was comparable in a c t i v i t y with an identical sample recrystallized in the absence of Tween 8o, if the Tween Re~eremites p. 56o.

55 s

VOJ~. 1 7 (I955)

12. VC CO\¥GILL

o...-o25

24 A----4

22

/

45 o o o -

/

o

0

N,

is

s5 m

.*~ D

/

m

_-,"

,4

-00

I

I

-4.0

-3.0

fa5

I

I

-2.0

-LO

Log of wgf.% Tween 80 Fig. "2. Correlation of a c t i v a t i o n of p h o s p h o r y l a s e a b y T w e e n 8o a n d c h a n g e of color of b r o m o t h y m o l blue b y Tween 80. A c t i v a t i o n of p h o s p h o r y l a s e a ; C h a n g e of color of b r o m o t h y m o l b l u e . . . . . 240

60

o_ o

o-..o. "o.

5

o40 ff o

160 ;lJ

. <(

/o

5" u~

~d

g

"13

""?,, . /

520

8O

na on

c~ _ _ 0

j . _ ~a ~ l -- 6 0

- 510

_~/ i

:-' I --

4. 0

J

I - 3 ,0

I

I

-20

Log of Moles of C D E A / l i t e r Fig. 3- Correlation of i n h i b i t i o n of p h o s p h o r y l a s e a b y C D E A a n d c h a n g e of color of b r o m o t h y m o l b l u e b y C D E A . I n h i b i t i o n of p h o s p h o r y l a s e a - - - - ; C h a n g e of color of b r o m o t h y m o l b l u e . . . . . D o t t e d circles indicate t h a t s a m p l e s were t u r b i d .

8o was diluted below its CMC value before the assay for a c t i v i t y (Table III). It m a y be noted that both the e n z y m e recrystallized in the presence and in the absence of Tween 8o were activated to the same extent by Tween 8o at concentrations above the CMC value in the assay mixture.

Re/erences p. 56o.

17 (I055) SURFACEACTIVE AGENTS AND RABI3IT MUSCLE PHOSPHORYLASE

VOL.

559

TABLE III ACTIVITY

OF

RABBIT

PHOSPHORYLASE IN

THE

RECRYSTALLIZED

ABSENCE

OF

T\VEEN

IN

THE

Assay without Tween ~o Conditions during recrvstallizalt~n*

Phosphoryh*se *in[Is a

Absence of Tween So Presence o f o o , ° , o Tween 80 •

*

~

I t.9 9.8

(a

hj

15.7 tl.9

Ratio a/(a ~ b)

0.76 0.82

PRESENCE

AND

80

Assay with o.o2 % Tween 8o Phosphorylase units a

20. 7 16.9

(a

h)

24.4 21.6

Ratio

a'(a

t,)

0.85 o.7S

See text for details of recrystallization.

DISCUSSION

Results of t h e e x p e r i m e n t s described above e x t e n d t h e o b s e r v a t i o n s t h a t certain enzymes are a c t i v a t e d or i n h i b i t e d b y SAA a n d t h a t t h e effects m a y be r e l a t e d to t h e f o r m a t i o n of micellar structures. If mieelle-formation is the critical change t h a t leads to p h o s p h o r y l a s e a c t i v a t i o n , the w a y b y which the micelles accomplish this was not revealed b y t h e p r e s e n t e x p e r i m e n t s . A c t i v a t i o n of p r o t y r o s i n a s e (ALLEN AND BODINE 1) a n d of g l u t a m i e acid d e c a r b o x y l a s e (HUGHES 1°) b y S A A are believed to involve rearr a n g e m e n t of t h e p r o t e i n to expose the enzyme m o i e t y in t h e first instance a n d r e m o v a l of an i n h i b i t o r in t h e second. N e i t h e r of these processes is in accord w i t h the results for a c t i v a t i o n of crystalline r a b b i t muscle p h o s p h o r y l a s e . A n o t h e r possibility would be t h a t the mieelle s t r u c t u r e m i g h t change t h e t h e r m o d y n a m i c e n v i r o n m e n t of t h e r e a c t i o n m i x t u r e . F o r example, t h e micelles m i g h t m a i n t a i n t h e r e l a t i v e l y huge p h o s p h o r y l a s e a n d glycogen molecules in positions favorable for r e p e a t e d interaction. The a b o v e results, however, give no i n d i c a t i o n of such a m e c h a n i s m , i.e., t h e e x t e n t of a c t i v a t i o n of r a b b i t muscle p h o s p h o r y l a s e b y Tween 80 does n o t v a r y with change of glycogen c o n c e n t r a t i o n of t h e assay m i x t u r e up to c o n c e n t r a t i o n s t h a t s a t u r a t e t h e enzyme. The results of Table I I I i n d i c a t e t h a t a c t i v a t i o n of the e n z y m e is not a p e r m a n e n t change in the e n z y m e molecule b u t persists only so long as t h e S A A is p r e s e n t in micelleforming c o n c e n t r a t i o n s in t h e a s s a y m i x t u r e . This p h e n o m e n o n s t a n d s in c o n t r a s t to t h e increased p h o s p h o r y l a s e a c t i v i t y of lobster muscle e x t r a c t s during storage 5. The l a t t e r increase in a c t i v i t y can be a c c e l e r a t e d b y a v a r i e t y of agents including surface active agents, b o t h ionic a n d nonionic. However, t h e change in crude lobster muscle e x t r a c t s a p p e a r s to be due to t h e conversion of an inactive p h o s p h o r y l a s e to p h o s p h o r y l a s e a. This change a p p e a r s to require o t h e r factors p r e s e n t in t h e crude e x t r a c t . Once the level of lobster p h o s p h o r y l a s e has risen, t h a t level is m a i n t a i n e d on purification a n d irrespect i v e of t h e presence or absence of surface active agents in t h e a s s a y m i x t u r e .

SUMMARY 1. Activity of rabbit nluscle phosphorylase a and phosphorylase b could be increased as much as two-fold by the addition of certain nonionic surface active agents• The concentrations of these agents required for activation of rabbit muscle phosphorylase coincide with the conceutrations required for aggregation of these agents into micelles. 2. Cationic and anionic surface active agents inhibit rabbit muscle phosphorylase. This inhibition also seems to be connected with the formation of micelles by these agents. lee/eremites p. 560.

56o

R. ~,v. CO~V(;ILL

VOL. 17 (~955)

RI;7.S t 7M I:: 1. 1.es activitds (le Ia p h o s p h o r y l a s e a et de la p h o s p h o r y l a s e b du 1hustle de lapin 1;euvent 6tre accrues j u s q u ' h deux fois par l'addition de certains agents tensioactifs non ioniques, lx~s concent r a t i o n s des d~tergents ndcessaires tt l'actiwltion de la t)hosphorylase du muscle de lapin coincident avec les c o n c e n t r a t i o n s pour lesquelles les ddtergents s'aggr6gent en micelles. 2. Les agents tensioactifs cationiques et aniolfiques i n h i b e n t la p h o s p h o r y l a s e du muscle de lapin. Cette inhibition semble 6galement 6tre en r a p p o r t ax'ec la formation de micelles par cos agents.

ZITS.XMMENFASSUNG i. Durch Hinzuf/igen von b e s t i m m t e n nichtionischen oberfl~tchenaktiven Sto~fen kolmte (lie Aktivitfit yon [ ' h o s p h o r y l a s e a u n d P h o s p h o r y l a s e b aus K a n i n c h e n m u s k e l n verdoppelt werden. Die fiir die Aktivierung yon K a n i n c h e n m u s k e l p h o s p h o r y l a s e ben0tigten Konzentrationen dieser St(fife s t i m m e n mit den K o n z e n t r a t i o n e n 6herein, welche notwendig sind, um diesc Stoffc zu Mizellen zusammenzuballen. 2. Kationische und anionische oberfltichenaktive Stoffe h e m m e n Kaninchennmsl~elphosphorylase. Diese H e m m u n g scheint gleichfalls mit der Mizellenbildung dicser Stoffe zusamnmnzuh/ingei1.

REFERENCES 1 THOMAS H..\LLEN AND JOSEPH H. BOmNU, Proc. Noc. Expll. BioL 3led., 49 (r942) 060. 2 C. F. CORI, G. T. CoRI AND A. A. GREEN, J. Biol. Chem., I 5 t (1943) 39a G. T. CORI AN1) C. F. CORI, J. Biol. Chem., i58 (1945) 32i. 4 M. g. CORRIN AND \~'ILLIAM D. IIARKINS, . / . . l m. (TheJ1~. Not., 09 (1947) 079. s R. \V. COWG,LL AND C. F. CORL ./. lJiol. Chem., (in press). 6 C. H. IrISKE AND Y. SUBBAROKV,,]. Hiol. Chem., 06 (I9"5) 375. 7 EMANUEL GONICK AND J . \\:. MCI{A1N, .]. :Ira. Chem. Noc., 69 0947) 334. A. A. GREEN AND G . T . CORI, J . Biol. Chem., I51 0943) 2i. " D. E. HUGHES, Biochem../., 45 ( [ 9 4 9 ) 3 ~ 5 • 10 D. E. HUGHES, BiocI~em. J., 40 U95 o) 231. 11 If. A. KREBS, Biochem. J., 42 (T948) V. le j . W. McBAIN, Colloid Scie~ce, D. C. Heath Co., 195o, Chap. 17. la MARTIN 1~. MATHEWS, l:ederatio~ Proc., [_' (I953) 243. ~4 FRANK \V. PU'rNAM, Adv. in ]'roleiJz Chem., 4 (1948) 79. Received February

I 4 t h , I95.5