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Activation of a growth factor by non-physiological means
204
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35226
A C T I V A T I O N OF A G R O W T H FACTOR BY N O N - P H Y S I O L O G I C A L MEANS
F R A N C I S W. S A Y R E * , M A U R I C E C. F I S H L E R " AND M I C H A E L E. J A Y K O * *
Kaiser Foundation Research Institute Richmond, Calif.,* and Lawrence Radiation Laboratory University of California Berkeley, Calif." (U.S.A.) (Received J a n u a r y i o t h , 1968)
SUMMARY
A protein growth factor, which shows growth-controlling properties in a number of living systems, has been found to undergo reversible activation (up to thirty-fold) of its biological activity. Biological activity and the effects of activation have been shown by promoting growth in nematode cultures, certain strains of tissue cultures, an obligate serum-requiring bacterium and by influencing the rate of growth of transplanted tumors in animals. The growth factor is present in serum and other tissues of mammals, including man and also in certain bacteria that have been examined. Activation can be accomplished by a variety of non-physiological means, as well as by a natural activator present in serum. A physiological inhibitor of activation has also been isolated from human serum. The non-physiological procedures for activation and deactivation mimic, at least in end result, the physiological activator and inhibitor. These non-physiological activation and deactivation procedures are reported here as a guide to understanding the chemistry of physiological control of the biological activity of circulating growth factor. Evidence is presented indicating activation and deactivation of growth factor to result from reversible conformational changes within the protein molecule.
INTRODUCTION
A protein present in the serum and other tissues of mammals, including man and also certain bacteria, has been shown to promote maturation and reproduction of the nematode Caenorhabditis briggsae in an otherwise chemically defined medium 1. The growth factor has been shown, by means of electrophoretic and chromatographic separations, to exist in multiple active forms. These forms have identical amino acid compositions and can be interconverted and are believed to be different-sized aggregates of a basic subunit 2. Each form of growth factor exists in activated and nonactivated states which are also interconvertiblel, 2. Maximal biological activity of most derived fractions could be demonstrated only after the protein was subjected to an activation process. Activation could be accomplished by each of several means Biochim. Biophys. Acta, 16o (1968) 204 216
GROWTH FACTOR ACTIVATION
205
including freezing in dilute solution, controlled heating, controlled dialysis, mixture with Ficoll and mixture with certain metal ions. Activation is a reversible process and enhances the biological activity for nematode maturation and reproduction at least 30-fold. Activated growth factor is effective for the culture of C. briggsae at concentrations as low as I0 #g per ml of defined medium. If the observed activation and deactivation of growth factor by non-physiological means have physiological meaning, it would be predicted that there would be physiological mechanisms to accomplish these purposes. In confirmation of this reasoning, a natural activator and a natural inhibitor of growth factor activity were recently isolated from the sera of man and several other mammals 3. Growth factor itself, in addition to its physiological modifiers, was found in these sera. Biological activity of activated growth factor has also been observed in culture of McCoY strain human epithelial cells4, 5, tumor-bearing animals2, 6, and cultures of parasitic nematodes 7, but C. briggsae provides by far the most convenient quantitative assay of growth factor and of its state of activation. This present study deals with the activation and deactivation of growth factor by non-physiological means. The results of these studies are being reported as a possible aid in understanding the physiological activation and deactivation mechanisms. It is believed that non-physiological activation procedures may mimic, at least in end result, the physiological activators in producing activation of growth factor. METHODS AND MATERIALS
Growth factor preparations were obtained from lamb liver by (NH4)2SO 4 precipitation followed by fractionation on hydroxylapatite columns as previously described2, 5. The protein content of the solutions was determined by the micro method of LOWRY et al.8. Ultracentrifugal characteristics were determined in the Spinco Model E analytical ultracentrifuge (Beckman Instruments, Palo Alto, California) using sedimentation velocity and ARCHIBALD approach-to-equilibrium methods 9. Viscosity measurements were made of growth factor solutions in chemically defined medium, both before and after activation by freezing. Measurements were done on 6 ml of solution in a Ubbelohde type viscometer with a water-efflux time at 3 °° of 298.76 sec (ref. IO). Biological activities were determined according to previously described methods1, 2. Three freshly hatched larvae of C. briggsae were inoculated into io-mm culture tubes containing 0.25 ml of defined medium and growth factor at the specified concentrations. The number of days required to go through a total life cycle and produce new larvae (F1 time) was determined for the test conditions. All materials were tested with and without activation by freezing in the chemically defined medium. The activity obtained after freeze activation in chemically defined medium was assumed to represent IOO % biological activity for comparative purposes. Activation of crude preparations of growth factor by freezing in the chemically defined medium and by controlled heating were first describedLll. Subsequent activation of purified growth factor fractions by these same procedures was reported 6. The rates of reaction of the sulfiaydryl groups of activated and non-activated Biochim. Biophys. Acta, 16o (1968) 2o4-216
206
F.w.
SAYRE, M. C. FISHLER, M. E. JAYKO
growth factor were determined by the method of BOYER1~, and by the method of ALEXANDERis using N-ethylmaleimide. Proteolytic digestion of activated and non-activated growth factor with trypsin was followed using a radiometer pH-stat and following the uptake of base as a function of time of digestion 14. The titratable groups of activated and non-activated growth factor were determined using a radiometer automatic titration apparatus. The optical rotatory dispersion of Ficoll-activated growth factor was studied using a Cary Mod~el 60 optical rotatory dispersion apparatus. RESULTS
Freeze activation The biological activity of freeze-activated growth factor compared to that of non-freeze-activated growth factor is shown in Fig. I over a range of growth factor concentrations. At low concentrations biological activity is not observed in nonactivated growth factor. However, as the concentration increases, activity can be
2,.50- ).o-o x w
~-.-~--
2O0-
.~O----ACTIVATEDBY FREEZING NON-ACTIVATED
o
z_ ~ 0
100-
g8 gf~_
.1" ~o ,oo
~o
pg G R O W T H
~o
~o
~ooo
FACTOR/ml ADDED
Fig. I. G r o w t h rate of C. briggsae cultures a t v a r i o u s c o n c e n t r a t i o n s of g r o w t h factor a d d e d to chemically defined m e d i u m . ~ - - - - - A , s u p p l e m e n t e d m e d i u m held at 4 °, o v e r n i g h t ; © - - O , s u p p l e m e n t e d m e d i u m frozen o v e r n i g h t a t - - 2 5 ° , a n d t h a w e d i m m e d i a t e l y before inoculation (freeze a c t i v a t e d ) . I n d i v i d u a l cultures c o n t a i n e d 0.25 m l of chemically defined m e d i u m cont a i n i n g g r o w t h factor at t h e c o n c e n t r a t i o n s indicated. C u l t u r e s were i n o c u l a t e d w i t h t h r e e l a r v a e each. T h e n u m b e r of d a y s required for a c o m p l e t e r e p r o d u c t i v e cycle (FI) were d e t e r m i n e d . T h e m a t u r a t i o n rate i n d e x is s h o w n as a f u n c t i o n of c o n c e n t r a t i o n .
obtained in the absence of activation. It can also be seen that above certain concentrations freeze activation does not give the full biological capability of the growth factor. This finding remains to be explained. The time required for complete freeze activation of growth factor was found to be a function of concentration of growth factor in the chemically defined medium (Table I). Activation occurs more rapidly at higher concentrations of protein and complete activation was obtained within 15 min. It is of interest to note that samples frozen in liquid N~ did not activate if thawed and tested immediately. If, however, they were warmed to --25 ° , activation proceeded normally in the times shown for --25 °. Conversely if activated growth factor was frozen in liquid Nz it remained Biochim. Biophys. Acta, 16o (1968) 2o4-216
207
GROWTH FACTOR ACTIVATION TABLE
I
TIME--CONCENTRATION
DEPENDENCE
OF F R E E Z E
ACTIVATION
G r o w t h f a c t o r , a d d e d t o defined media at varying c o n c e n t r a t i o n s , w a s f r o z e n for t h e indicated t i m e s a t - - 2 5 ° . T u b e s w e r e t h a w e d and inoculated by t h e u s u a l a s s a y p r o c e d u r e . R e s u l t s are s h o w n as % maximal activity for t h e c o n c e n t r a t i o n s s h o w n , i.e., percent activation.
Time of freezing (h)
Growth factor (tzg/ml)
Percent activation by freezing 25o
5o
37.5
25
I2.5
O.2 5
IOO
O
O
O
O
0.50
ioo
7o
io
o
o
I.O
I00
IO0
80
50
o
2,o
I0O
I00
IO0
IO0
IO
4.o
IOO
IOO
IOO
IOO
IO0
IOO
IOO
IOO
IOO
IOO
8
9
64.0 C o n t r o l F~ t i m e (days)
4
4½
6½
activated when thawed quickly in a water bath and assayed for biological activity. The reversible nature of activation by freezing is shown in Table II. Aliquots of growth factor in defined medium were activated by the normal freeze-activation procedure and held for varying times at 37 ° . At the appropriate time, these samples were tested without refreezing and after refreezing to determine the residual activation and also the potential activity after complete activation by refreezing. At 37 °, activation (by freezing) begins to decay and by 24 h has completely decayed. Refreezing, TABLE
II
GROWTH FACTOR: FREEZE-ACTIVATION
DECAY--REACTIVATION
S a m p l e s , c o n t a i n i n g 7 5 / z g / m l o f g r o w t h f a c t o r i n chemically defined m e d i u m , w e r e f r o z e n ( -- 25 °) o v e r n i g h t for r o u t i n e a c t i v a t i o n . S a m p l e s w e r e w i t h d r a w n a t a p p r o p r i a t e t i m e s a n d s t o r e d for t h e i n d i c a t e d t i m e s a t 37 ° . A s s a y t u b e s w e r e i n o c u l a t e d w i t h l a r v a e a f t e r o v e r n i g h t s t o r a g e a t 4 ° ( n o t r e f r o z e n ) , or a f t e r o v e r n i g h t h o l d i n g a t - - 2 5 ° (freeze activation repeated). Similar decay o f f r e e z e activation and reactivation by r e f r e e z i n g w e r e a l s o s h o w n a t 3 o°, a n d a t 20 ° ; (see t e x t ) . T h e m a t u r a t i o n t i m e s s h o w n are t h e a v e r a g e o f d u p l i c a t e a s s a y s .
Time at 37 ° after original activation (h) o (control) I 2 3 8 24
Maturation time (days) Not refrozen Refrozen 7.5
ii n.m.*
6.5 6.75 6.75 6 6 6.5 °
* Non-maturing
a t 2 i days.
7 7 8.25
however, restores the full biological activity of the material, thus establishing the reversible nature of the activation process when accomplished by freeze activation. Similar decay of freeze activation and reactivation by refreezing were also demonstrated at 3 °o and at 20 °. Decay of activation was slower and some residual activation remained after 48 h at 3 °0 and after 3 days at 20 °. Complete activation could, in every case, be restored by refreezing. Biochim. Biophys. Acta, 16o (1968) 2 o 4 - 2 1 6
208
F. W. S A Y R E , M. C. F I S H L E R , M. E. J A Y K O
TABLE III EFFECT
OF H E A T I N G
ON G R O W T H F A C T O R A C T I V I T Y
G r o w t h f a c t o r s o l u t i o n s (I m g / m l ) w e r e t r e a t e d as i n d i c a t e d . S u i t a b l e a l i q u o t s w e r e a d d e d t o d e f i n e d m e d i u m t o g i v e 5 ° / ~ g / m l g r o w t h f a c t o r a n d t e s t e d for b i o l o g i c a l a c t i v i t y .
Conditions
4 °, o v e r n i g h t 37 ° , o v e r n i g h t 53 °, 5 m i n 7 o°, 5 m i n IO°°, 5 m i n Autoclaved, t5 min
Mataralion time (days) Unfrozen"
Frozen**
n.m.*** 5-5 5.75 n.m. n.m. n.m.
4-5 5 5 n.m. n.m. n.m.
Unfrozen (not activating conditions). "" A f t e r f r e e z i n g o v e r n i g h t a t - - 2 5 ° ( a c t i v a t i n g c o n d i t i o n s ) . "*" n . m . , n o n - m a t u r i n g a t 21 d a y s . M a t u r a t i o n t i m e s are a v e r a g e of d u p l i c a t e a s s a y s .
Heat activation
The effect of heating on activity of growth factor is shown in Table III. Incubation of growth factor alone at 37 ° overnight produces essentially complete activation ; subsequent freeze activation in defined medium produces only a slight increase in activity under these conditions. At 53 ° for 5 min nearly complete activation of growth factor is observed. Subsequent freeze activation in defined medium brings this to full activity. 7 °° for 5 rain irreversibly destroys all biological activity of the growth factor TABLE IV HEAT ACTIVATION--CONCENTRATION DEPI!NDENCE S a m p l e s o f g r o w t h f a c t o r w e r e h e a t e d a t t h e i n d i c a t e d c o n c e n t r a t i o n s a n d t e m p e r a t u r e s for 2.5 m i n . T u b e s w e r e p r e h e a t e d t o t e m p e r a t u r e b e f o r e a d d i t i o n of g r o w t h f a c t o r s o l u t i o n s . A f t e r h e a t i n g s a m p l e s w e r e c h i l l e d i n a n ice b a t h . A n e q u a l a m o u n t o f d e f i n e d m e d i u m w a s a d d e d a n d s a m p l e s w e r e d i v i d e d i n t o f o u r t u b e s . T w o w e r e t e s t e d a f t e r s t a n d i n g o v e r n i g h t a t 4 ° (nona c t i v a t i n g ) , a n d t h e o t h e r t w o w e r e f r o z e n (for a c t i v a t i o n ) . All s a m p l e s w e r e i n o c u l a t e d a t t h e s a m e t i m e a n d f o l l o w e d b y t h e s t a n d a r d a s s a y p r o c e d u r e . T h e t i m e s for r e p r o d u c t i o n are t h e a v e r a g e of d u p l i c a t e a s s a y s .
Temp.
Maturation time (days) Activation level Test level
4001~g/ml" 20o #g/ml*
zoo #g/ml* 50 #g/ml*
50 I~g/ml * 25 t~g/ml"
Unfrozen
Frozen
Unfrozen Frozen
Unfrozen Frozen
5 °0 51° 520 53 ° 54 ° 55 °
n.m. n.m. 9.5 5.5 5.5 5
4.25 4 4 4.5 4.5 4-5
n.m. n.m. n.m. n.m. 9 8
4.5 4-5 5 4.5 5.5 5.5
n.m. n.m. n.m. n.m. n.m. n.m.
5.5 7 5.5 6 5-5 6
N o h e a t i n g (control)
n.m.
4-5
n.m.
5
n.m.
6
* Growth factor concentration.
Biochim. Biophys. Acta, 16o (1968) 2 o 4 - 2 1 6
209
GROWTH FACTOR ACTIVATION
either with or without freeze activation. The same irreversible destruction of growth factor activity was found after heating at IOO° for 5 rain or after autoclaving. It was thus shown that heating can activate the growth factor or completely destroy all biological activity in an irreversible manner depending on concentration, temperature, and time of exposure. Heat activation was found to be highly dependent upon concentration at various temperatures (Table IV). Under these conditions, partial activation of growth factor by controlled heating can be brought to full activation by subsequent freezing in chemically defined medium. This suggests that the two processes are related. Heat activation at the body temperature of mammals was also found to be highly concentration dependent (Table V). No evidence of heat activation (37 °) was obtained at
TABLE V ACTIVATION BY HEATING (CONCENTRATION DEPENDENCE) Samples of g r o w t h factor, at the indicated concentrations, were held at 37 ° for 24 h (Column i). The heated g r o w t h factor was bioassayed at 75/~g/ml in defined m e d i u m - - w i t h o u t activation (4 ° overnight in defined medium) and after activation b y freezing (--25 ° overnight in defined medium). M a t u r a t i o n times s h o w n are the average of triplicate assays for each point, n.m., non-maturing.
Growth factor concentration
Maturation time (days)
Heated (l~g/ml)
Tested (l~g/ml)
Unfrozen Frozen
3 oo° 15oo 75 ° 45 ° 15° Unheated
75 75 75 75 75 75
6.5 n.m. n.m. n.m. n.m. n.m.
5.5 5.5 5.5 5.5 5.5 5.5
low concentrations of growth factor, even though these were still considerably above physiological concentrations of growth factor. The average physiological concentration of circulating growth factor is about 70/~g/ml. This indicates that physiological activation is probably not accomplished by controlled heating since relatively high concentrations of growth factor are required for activation at physiological temper atures. Mixture with metalic cations
Certain divalent cations have been shown to activate growth factor while other metalic ions are without effect under the conditions tested (Table VI). The relative concentrations of metal to protein are important. Magnesium, calcium, manganese and cadmium showed no activation of growth factor under these conditions, however, freeze activation occurred normally after exposure to these (metal ions). Manganese ion showed a possible slight stimulation of activity after freeze activation. The sulfhydryl-blocking reagents were shown to destroy biological activity of growth factor. Another group of compounds (EDTA, oxidized and reduced glutathione and dithioBiochim. Biophys. Acta, 16o (1968) 2o4-2x6
210
F. W. SAYRE, M. C. FISHLER, M. E. JAYKO
T A B L E VI METAL
ION
ACTIVATION
All i n c u b a t i o n s were for i 8 h a t p r o t e i n c o n c e n t r a t i o n s of i o o o / ~ g / m l a n d a g e n t a t i mM (unless o t h e r w i s e i n d i c a t e d ) a t 4°; b i o a s s a y s were done w i t h a n d w i t h o u t freeze a c t i v a t i o n a t i o o / ~ g p r o t e i n / m l . E s s e n t i a l l y t h e s a m e r e s u l t s were o b t a i n e d a t g r o w t h fa c t or c o n c e n t r a t i o n s of 25 ~ g / m l . The s a m p l e freeze a c t i v a t e d in defined m e d i u m , w i t h o u t a n y a d d i t i o n , w a s a c c e p t e d as t he i oo % s t a n d a r d of a c t i v i t y for t h e s e e x p e r i m e n t s .
Agent added
% Activation Without freeze activation
None MgCI~ CaC12 MnCI 2 CaC12 HgC12 N-Ethylmaleimide EDTA G l u t a t h i o n e (reduced) G l u t a t h i o n e (oxidized) Dithiothreitol CoCI~ Fe(NH4) 2SO~ CuSO 1 NiC12 ZnC12 (o.oI M) ZnC12 (o.ooi M)
o
o o o o o o o o o o 86 82 69 ioo 7o o
Freeze activated*
I oo * ~
Ioo ioo 13 ° ioo o o 9o Ioo ioo I oo Ioo Ioo 72 9o 48 ioo
* After freezing in defined m e d i u m to give n o r m a l a c t i v a t i o n conditions. ** 4,5 d a y g e n e r a t i o n time. (MRI = I / F j x i 0 0 0 = 222.2) IOO~0a c t i v a t i o n .
threitol) showed essentially no effect upon the state of activation of growth factor or upon the ability of growth factor to freeze activate (except for E D T A which showed slight inhibition of activation upon freezing). The next group of metal ions (cobalt, iron, copper, nickel, and zinc) showed some activation of growth factor under the conditions tested. It is of interest to note that cobalt and Fe 2÷ showed incomplete activation of growth factor, yet did not prevent subsequent complete activation by the freeze-activation procedure. Nickel and zinc are of interest in that they showed activation of growth factor, however, a slight depression of the activity (or activation) was observed after freeze activation. ZnC12, at a lower concentration, showed no activation of growth factor but permitted complete activation by freezing. Cu ~+ is of special interest in that it shows different effects, depending on its concentration (Table VII): (I) it shows no effect, (2) can facilitate freeze activation, (3) can activate b y itself, (4) can completely block all activation. At low concentrations, Cu 2+ shows no effect upon the state of activation and shows no interference in the freeze-activation process. At o.I mM, Cu *+ produces a 53 % activation without freeze activation, however, upon freezing, activation becomes complete. At I mM, where Cu 2+ gives an 8o °/o activation, upon freezing only 55 ~/o of the control biological activity was found. At o.oi M, Cu 2+ not only did not activate but prevented the freeze activation. Inhibition of growth factor activity at high concentrations of Cu 2+ is likely Biochim. Biophys. Acla, 16o (1968) 2o4--216
GROWTH FACTOR ACTIVATION TABLE
211
VII
C u 2+ ACTIVATION OF GROWTH FACTOR E x p o s u r e o f p r o t e i n w a s a t IOOO/~g p r o t e i n / m l a t t h e C u 2+ c o n c e n t r a t i o n s s h o w n . B i o a s s a y s w e r e d o n e a t i o o k t g / m l p r o t e i n a n d a t 2 5 / ~ g / m l p r o t e i n . T h e f r e e z e - a c t i v a t e d s a m p l e , w i t h o u t C u 2+, was accepted as the lOO% standard of activity for these experiments.
CuSO 4 conch. % Activation at exposure (raM) Without Freeze freeze activated activation O IO 1 o.I O.Ol o.ooi
O O 80 53 o o
IOO O 55 IOO ioo ioo
due to interaction of copper with the sulthydryl groups of growth factor which are essential for its biological activity 15. Mixture with Ficoll--a synthetic polymer of sucrose distributed by Pharmacia Fine Chemicals--has been shown to activate growth factor. This activation has been discussed by BUECHER,HANSEN AND YARWOOD16. It has since been found that the concentrations of Ficoll required for activation vary with concentrations of growth factor protein and somewhat from preparation to preparation of growth factor. Ficoll is reported to have a molecular weight of approx. 400 ooo. It was designed for densitygradient centrifugation studies and has been presumed to be chemically inert. The only reactive chemical groups of Ficoll presumably are aliphatic hydroxyl groups (about 38 %). We found however that preparations of Ficoll in solution give a crystalline reaction product with 2,4-dinitrophenyihydrazine. This could indicate the presence of either an aldehyde or keto group which theoretically should not be present and may be so only as a result of partial decomposition or impurities of the Ficoll. The parent compound, sucrose, which has the same chemical groups as Ficoll, blocked activation of the growth factor by the freeze-activation process at concentrations comparable to Ficoll on a weight basis. As yet, the mechanism of activation by Ficoll remains unexplained. Activation by mixture with Ficoll does, however, increase chemical reactivity of the sulthydryl groups of growth factor in the same way as other activation procedures 15. Although a great deal is known of the interactions of protein molecules with cationic and anionic molecules 17 very little is known of the interaction of nonionic molecules, such as Ficoll, with proteins. Activation of growth factor by other non-physiological means has been observed under various conditions but has been inconsistent. Low ionic strength and pH of buffers have been observed to activate growth factor under certain conditions and have been discussed by BUECHER,HANSEN AND YARWOOD16. Attempts to study these conditions of activation in detail have shown them to be inconsistent. Likewise, spontaneous activation occasionally occurs upon prolonged storage, but this too is inconsistent, rare and unexplainable on the basis of the known chemistry of growth factor. Biochim. Biophys. Acta, 16o (1968) 2 o 4 - 2 1 6
212
F. W. SAYRE, M. C. FISHLER, M. E. JAYKO
Evidence of structural changes upon activation Several lines of evidence indicate that structural changes occur during the activation of growth factor, irrespective of the activation procedure. Several of the activation procedures show overlapping in the nature of the evidence for conformational changes following activation of the growth factor. Any one of these indications alone would not be sufficient to establish a conformational change related to the activation process. However, when they are all considered together, they make fairly convincing evidence that critical structural changes are responsible for the activation of growth factor. YANKEELOVAND KOSHLAND18 have evaluated the criteria for conformational changes and pointed out the strength of diverse and overlapping evidence for conformational changes. The specific evidence of a structural change in the protein molecule with activation of growth factor is : Differential reactivity of sulJhydryl groups. Changes in the chemical reactivity of the sulfhydryl groups of growth factor have been observed after activation by heating and by mixture with Ficol115. The maximal number of sulfhydryl groups reacting with
0
"
001
o
i
NONACTIVAT
__./ ~
{
003
m
004
o
005
ACTIVATED
•
BY FREEZrNG
X 5o uJ
006 007
I IO
I 20 MINUTES
I 30
I
40
--
008200
~o
,~o
WAVELENGTH, MILLIMICRONS
~o
Fig. 2. Time course of digestion of g r o w t h factor in defined m e d i u m (e~--O) and g r o w t h factor freeze activated in defined m e d i u m (@ @!. Digestion was followed by base u p t a k e with a R a d i o m e t e r t i t r a t o r used as p H star. The p H was adjusted to 7.8o before the addition of t r y p s i n and digestion was allowed to proceed. Values are corrected so t h a t each point is due only to H + liberated by digestion. Samples contained g r o w t h factor, 2Io #g in defined m e d i u m and 20/tg t r y p s i n in a final volume of i .o ml. The samples were identical except t h a t one was activated by freezing overnight at --25 °. Only the first 35 rain are shown, b u t total base u p t a k e was the same for the two samples after 24 h. Similar results were obtained with Ficoll activated g r o w t h factor. Fig. 3. 0 - - 0 , optical r o t a t o r y dispersion of g r o w t h factor (non-activated); Q - - © , same of g r o w t h factor activated with Ficoll at 5 % (corrected for Ficoll rotation). The samples were growth factor at i mg/ml, in o.i M p o t a s s i u m p h o s p h a t e buffer; and g r o w t h factor at i m g / m l activated in o.I M p o t a s s i u m p h o s p h a t e buffer containing 5 % Ficoll. The p H of b o t h buffer solutions was 7.0. The curves are corrected for buffer and for buffer plus 5 % Ficoll so t h a t the values s h o w n are the r o t a t i o n s observed for the protein solutions a]one, non-activated, and after activation b y m i x t u r e with Ficoll. Activation by Ficoll and non-activation of the control were confirmed b y bioassay of aliquots of each sample. These determinations were made on a Cary-6o optical r o t a t o r y dispersion a p p a r a t u s b y Dr. M. JAYKOat the University of California.
Biochim. Biophys. Acta, 15 ° (1968) 2o4-216
GROWTH FACTOR ACTIVATION
213
p-chloromercuribenzoate was found to be the same for activated and non-activated growth factor while their spatial arrangement appears to be different as evidenced by their different rates of chemical reaction. Susceptibility to trypsin digestion. The time course of digestion of non-activated growth factor is a smooth one, whereas, digestion of growth factor (freeze activated) occurs in discrete steps (Fig. 2). The total number of bonds split by trypsin after 24 h digestion is the same for activated and for non-activated growth control factor. Similar results were obtained with growth factor activated by Cu 2+ and by mixture with Ficoll. ]'he step-wise digestion of activated growth factor by trypsin indicates activated growth factor to be a more highly ordered structure than non-activated growth factor. The immediate splitting of one peptide bond per 21 ooo g of growth factor protein indicates that one lysine (or arginine) group is more susceptible to tryptic digestion in activated growth factor than in non-activated growth factor, probably by virtue of its greater accessibility to trypsin. The same findings after activation of growth factor by three different procedures suggest similar structural changes in this region of the molecule. Titratable groups. Non-activated growth factor shows 14 groups per 21 ooo g of protein titratable at the pK of e-aminolysine (Table VIII). Growth factor activated by freeze activation shows only 5 of these groups. Amino acid analysis showed 14 lysine TABLE VIII T I T R A T A B L E G R O U P S OF G R O W T H F A C T O R
T i t r a t i o n s w e r e done w i t h a R a d i o m e t e r p H s t a t in defined m e d i u m . All v a l u e s are c orre c t e d for defined m e d i u m , so are n e t for protein.
p H range
7 . 0 - 8.0 8 . 0 - 9.0 9.0 io,o io.o-ii.o I I.O--I2,0
Titrated groups (equiv/2i ooo g protein) Non-activated
d c!ivated
4.4 I2.2 25. 9 13. 9 I6. 3
0.8 0.8 2. 5 4.9 3.9
groups per 21 ooo molecular weight. This would suggest all e-aminolysine groups are available in the non-activated condition and 9 are folded in after activation 19. These findings also provide good substantiation of the amino acid composition and of the 21 ooo molecular weight basic unit. Optical rotatory dispersion. The optical rotatory dispersion of growth factor and of Ficoll activated growth factor are shown in Fig. 3. Non-activated growth factor shows minimum Cotton Effect at 238 m/~ which shifts to approx. 235 m# after activation with Ficoll z0. There is also a pronounced decrease in levorotation, indicating a more ordered structure zl. A crossing of the zero axis of rotation by the activated growth factor (corrected for Ficoll rotation) furthermore indicates a conformational change within the protein. Since the optical rotatory dispersion curves were determined only after activation with Ficoll, the optical rotatory dispersion studies can only be used Biochim. Biophys. Acta, i 6 o (1968) 2o4-216
214
F.w.
SAYRE, M. C. FISHLER, M. E. JAYKO
as confirmatory evidence of the other indications of conformational changes which occur with activation. Viscosity changes with activation. In a typical experiment, a solution of growth factor at 2oo #g/ml in chemically defined medium was found to have an efflux time of 315.14 sec from the capillary of a Ubbelohde viscometer, corresponding to a value of o.84826 centistokes. After activation by freezing overnight, another aliquot of this same solution was found to have an efflux time of 323.o 9 sec, corresponding to o.86966 centistokes. The first three readings of the efflux time of activated growth factor were in good agreement, but the effiux time of the fourth reading was found to be 315.57 sec or essentially the same as the non-activated growth factor. This reading remained constant. Control bioassays indicated that the growth factor had been activated when the original viscometric measurements were made and that after passage through the viscometer three times, the material was no longer activated, but could be reactivated upon refreezing. Thus, the shearing action of passing activated growth factor through the capillary tube of the viscometer resulted in its biological deactivation as well as a return of the efflux time to that of nonactivated growth factor. Interaction with physiological inhibitor. Physiological inhibitor of growth factor activity has recently been isolated from human serum and shown to interact with growth factor; this interaction was shown by ultracentrifugal analysis and by measured changes in the chemical reactivity of the sulthydryl groups of growth factor. The inhibitor of growth factor activity is a new protein which reacts quantitatively with growth factor and suppresses its biological activity in the nematode maturation assay ~. The suppressive effect of natural inhibitor on growth factor activity can be overridden by natural activator (in higher concentrations) or by Ficoll (a powerful non-physiological activator).
DISCUSSION
Similar quantitative changes in the biological activity of growth factor have been observed in the nematode maturation assay following activation by widely varying procedures. There has been evidence of a conformational change in the protein following each of the activation procedures 2=. For technical reasons it has been impractical to apply all criteria cited for conformational changes to all activation procedures. It has, however, been possible to detect sufficient overlap in these criteria to provide evidence of similar structural changes following different activation procedures. For example: (I) Similar changes in the susceptibility of growth factor to tryptic digestion were found after activation by freezing in defined medium, controlled heating, mixture with Ficoll and mixture with Cu 2+. (2) Similar changes in the chemical reactivity of the sulthydryl groups of growth factor were found after activation by controlled heating or mixture with Ficoll; sulihydryl groups are of special interest as they have been shown to be essential to biological activity. (3) Similar changes in the titratable groups of growth factor have been observed following activation by freezing, mixture with Ficoll, and mixture with Cu =+; this shows different degrees of exposure of specific chemical groups of growth factor in the activated and non-activated condition. (4) Changes in the optical rotatory dispersion have been observed following activation with Ficoll. This overlapping evidence of conformational Biochitn. Biophvs. Acta, 16o (I968) 2o4-216
GROWTH FACTOR ACTIVATION
215
changes following activation by different procedures indicates that critical structural changes must occur during activation by any of these procedures. Reversibility of most of the activation procedures described further substantiates the idea of a reversible conformational change within the protein molecule and its relationship to the activation mechanism. The finding that partial activation by one procedure can be brought to full activation by a second and different activating procedure indicates that similarities exist in the different activation procedures. All of these evidences considered together suggest that critical conformational or structural changes occur during activation which correlate with changes in the observed biological activity of growth factor. The various methods of activation described above have resulted in increased or modified biological activity in other systems such as cell culture in vitro, bacterial growth, and retardation of growth of transplanted tumors. Activation by mixture with Ficoll and by freezing in chemically defined medium (or nucleotides) has been observed in all biological systems studied thus far, and has been found to alter significantly the biological activity of growth factor in each system. Activation by controlled heating has been demonstrated in all biological systems studied except in the growth of transplantable tumors (where it has not been tried). Cu 2+ and other metal ion activation have been studied only with the nematode maturation assay. Activation of a number of enzymes by similar means has been observed for some time. One of the earliest recognitions of this phenomenon can be credited to HOFSTEE23 and came from his studies on urease activity. Reversible activation of urease was described by controlled heating and by freezing under certain conditions. More recent work indicates that these activations are the result of conformational changes within the protein molecule. Recent specific examples of conformational changes accompanying altered biological activity (i.e., activation and deactivation) have been clearly shown for: (I) phosphoglucomutase24; (2) alkaline phosphatase from Escherichia c01i25; and (3) dihydrofolate reductase from L-I2IO cells26. In all of these examples, limited conformational changes lead to activation of the enzyme--usually reversible. More extensive conformational changes, often by the same agents, result in irreversible inactivation of biological activity. These findings parallel and overlap in many ways the findings with growth factor and suggest that such regulatory mechanisms are far more common for regulation of biological processes than had been heretofore recognized. The discovery of the physiological activator and inhibitor as well as growth factor in the sera of man and other mammals is significant from several points of view. First, it provides a physiological control mechanism for the above-described conformational changes in the growth factor. Second, it gives further meaning to the work described herein on nonphysiological activation procedures. Third, it provides a start in the understanding of a newly recognized homeostatic mechanism possibly concerned with cellular growth in mammals.
ACKNOWLEDGMENT
This investigation was supported in part by Grant AM-o6oo4 from the National Institutes of Health, U.S. Public Health Service. Biochim. Biophys. Acta, 16o (1968) 2o4-216
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F. W. SAYRE, M. C. FISHLER, M. E. JAYKO
REFERENCES i F.W. SAYRE, E. HANSEN AND E. A. YARWOOD, Exptl. Parasitol., 13 (1963) 98. 2 F. W. SAYRE, R. T. LEE, R. P. SANDMAN AND G. PEREZ-MENDEZ, Arch. Bioehem. Biophys., 118 (1967) 58 . 3 F. W. SAYRE AND M. C. FISHLER, s u b m i t t e d for publication. 4 T. J. STARR, M. POLLARD, Y. TANAMI AND R. MOORE, Texas Rep. Biol. Med., 18 (196o) .5Ol. 5 F. W. SAYRE, E. NANSEN, T. J. STARR AND E. A. YARWOOD, Nature, 19o (I961) 1116. 6 F. W. SAYRE, E. I-{ANSEN, P. BARNARD AND E. A. YAR~VOOD, Federation Proc., 21 (1962) 238. 7 P. H. SILVERMAN, E. L. HANSEN, E. BUECHER, J. Parisitol., 51 (1965) 46. 8 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND m. J. RANDALL, jr. Biol. Chem., I93 (1951) 265 . 9 H. ~(-. SCHACHMAN, Ultracentrifugation in Biochemistry, Academic Press, New York, 1959, p. 63 and 181. lO A. M. KRAGH, A Laboratory Manual of Analytical Methods of Protein Chemistry, Vol. 3, Perg a m o n Press, L o n d o n 1961, p. 173. l 1 E. HANSEN, F. ~V. SAYRE AND E. A. YARWOOD, Experzentia, 17 (1961) 32. 12 P. D. BOYER, J. Am. Chem. Soc., 76 (1954) 4331. 13 N. M. ALEXANDER, Anal. Chem., 3 ° (1958) 1292. 14 C. F. JACOBSEN, J. LEONIS, K. LINDERSTROM-LANC AND M. OTTESEN, Methods of Biochemical Analysis, Vol. 4, Interscience, New York, 1957, p. i. 15 F. W. SAYRE, M. C. FISHLER, G. HIJMPHREYS .aND M. E. JAYKO, Biochim. Biophys. Acta, 16o (I968) 63. 16 E. J. BUECHER, E. HANSEN .aND E. A. YARWOOD, Proc. Soc. Exptl. Biol. Med., I2I (1966) 39o. I 7 P. BERNFELD, B. J. BERKELEY AND R. E. BIEBER, Arch. Biochem. Biophys., II1 (1965) 31. 18 J. A. YANKEELOV AND D. E. KOSHLAND, JR., J. Biol. Chem., 240 (1965) 1593. I9 C. TANFORD, Physical Chemistry of Macromolecules, J o h n \Viley and Sons, New York, I96t, p. 564 . 20 C. TANFORD, Physical Chemistry, of l~Iacromolecsles, J o h n Wiley and Sons, New York, 196I, p. 119. 21 \V. F. HARRINGTON, R. JOSEPHS AND D. M. SEGAL, Ann. Rev. Biochem., 35 (1966) 599. 22 S. N. TIMASHEFF AND M. J. GORBUNOFF, Ann. Rev. Biochem., 36 (1967) 13. 23 13. J. HOFSTEE, J. Gen. Physiol., 32 (1948) 339. 24 V. BOCCHINI, M. R. ALIOTO AND V. A. NAJJAR, Biochemistry, 6 (1967) 3242. 25 J. A. REYNOLDS AND M. J. SCHLESINGER, Biochemistry, 6 (1967) 3552. 26 P. REYS AND F. M. HUENNEKENS, Biochemistry, 6 (I967) 3519.
Biochim. Biophys. Acta, i6o (I968) 2o4-216
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 35226
A C T I V A T I O N OF A G R O W T H FACTOR BY N O N - P H Y S I O L O G I C A L MEANS
F R A N C I S W. S A Y R E * , M A U R I C E C. F I S H L E R " AND M I C H A E L E. J A Y K O * *
Kaiser Foundation Research Institute Richmond, Calif.,* and Lawrence Radiation Laboratory University of California Berkeley, Calif." (U.S.A.) (Received J a n u a r y i o t h , 1968)
SUMMARY
A protein growth factor, which shows growth-controlling properties in a number of living systems, has been found to undergo reversible activation (up to thirty-fold) of its biological activity. Biological activity and the effects of activation have been shown by promoting growth in nematode cultures, certain strains of tissue cultures, an obligate serum-requiring bacterium and by influencing the rate of growth of transplanted tumors in animals. The growth factor is present in serum and other tissues of mammals, including man and also in certain bacteria that have been examined. Activation can be accomplished by a variety of non-physiological means, as well as by a natural activator present in serum. A physiological inhibitor of activation has also been isolated from human serum. The non-physiological procedures for activation and deactivation mimic, at least in end result, the physiological activator and inhibitor. These non-physiological activation and deactivation procedures are reported here as a guide to understanding the chemistry of physiological control of the biological activity of circulating growth factor. Evidence is presented indicating activation and deactivation of growth factor to result from reversible conformational changes within the protein molecule.
INTRODUCTION
A protein present in the serum and other tissues of mammals, including man and also certain bacteria, has been shown to promote maturation and reproduction of the nematode Caenorhabditis briggsae in an otherwise chemically defined medium 1. The growth factor has been shown, by means of electrophoretic and chromatographic separations, to exist in multiple active forms. These forms have identical amino acid compositions and can be interconverted and are believed to be different-sized aggregates of a basic subunit 2. Each form of growth factor exists in activated and nonactivated states which are also interconvertiblel, 2. Maximal biological activity of most derived fractions could be demonstrated only after the protein was subjected to an activation process. Activation could be accomplished by each of several means Biochim. Biophys. Acta, 16o (1968) 204 216
GROWTH FACTOR ACTIVATION
205
including freezing in dilute solution, controlled heating, controlled dialysis, mixture with Ficoll and mixture with certain metal ions. Activation is a reversible process and enhances the biological activity for nematode maturation and reproduction at least 30-fold. Activated growth factor is effective for the culture of C. briggsae at concentrations as low as I0 #g per ml of defined medium. If the observed activation and deactivation of growth factor by non-physiological means have physiological meaning, it would be predicted that there would be physiological mechanisms to accomplish these purposes. In confirmation of this reasoning, a natural activator and a natural inhibitor of growth factor activity were recently isolated from the sera of man and several other mammals 3. Growth factor itself, in addition to its physiological modifiers, was found in these sera. Biological activity of activated growth factor has also been observed in culture of McCoY strain human epithelial cells4, 5, tumor-bearing animals2, 6, and cultures of parasitic nematodes 7, but C. briggsae provides by far the most convenient quantitative assay of growth factor and of its state of activation. This present study deals with the activation and deactivation of growth factor by non-physiological means. The results of these studies are being reported as a possible aid in understanding the physiological activation and deactivation mechanisms. It is believed that non-physiological activation procedures may mimic, at least in end result, the physiological activators in producing activation of growth factor. METHODS AND MATERIALS
Growth factor preparations were obtained from lamb liver by (NH4)2SO 4 precipitation followed by fractionation on hydroxylapatite columns as previously described2, 5. The protein content of the solutions was determined by the micro method of LOWRY et al.8. Ultracentrifugal characteristics were determined in the Spinco Model E analytical ultracentrifuge (Beckman Instruments, Palo Alto, California) using sedimentation velocity and ARCHIBALD approach-to-equilibrium methods 9. Viscosity measurements were made of growth factor solutions in chemically defined medium, both before and after activation by freezing. Measurements were done on 6 ml of solution in a Ubbelohde type viscometer with a water-efflux time at 3 °° of 298.76 sec (ref. IO). Biological activities were determined according to previously described methods1, 2. Three freshly hatched larvae of C. briggsae were inoculated into io-mm culture tubes containing 0.25 ml of defined medium and growth factor at the specified concentrations. The number of days required to go through a total life cycle and produce new larvae (F1 time) was determined for the test conditions. All materials were tested with and without activation by freezing in the chemically defined medium. The activity obtained after freeze activation in chemically defined medium was assumed to represent IOO % biological activity for comparative purposes. Activation of crude preparations of growth factor by freezing in the chemically defined medium and by controlled heating were first describedLll. Subsequent activation of purified growth factor fractions by these same procedures was reported 6. The rates of reaction of the sulfiaydryl groups of activated and non-activated Biochim. Biophys. Acta, 16o (1968) 2o4-216
206
F.w.
SAYRE, M. C. FISHLER, M. E. JAYKO
growth factor were determined by the method of BOYER1~, and by the method of ALEXANDERis using N-ethylmaleimide. Proteolytic digestion of activated and non-activated growth factor with trypsin was followed using a radiometer pH-stat and following the uptake of base as a function of time of digestion 14. The titratable groups of activated and non-activated growth factor were determined using a radiometer automatic titration apparatus. The optical rotatory dispersion of Ficoll-activated growth factor was studied using a Cary Mod~el 60 optical rotatory dispersion apparatus. RESULTS
Freeze activation The biological activity of freeze-activated growth factor compared to that of non-freeze-activated growth factor is shown in Fig. I over a range of growth factor concentrations. At low concentrations biological activity is not observed in nonactivated growth factor. However, as the concentration increases, activity can be
2,.50- ).o-o x w
~-.-~--
2O0-
.~O----ACTIVATEDBY FREEZING NON-ACTIVATED
o
z_ ~ 0
100-
g8 gf~_
.1" ~o ,oo
~o
pg G R O W T H
~o
~o
~ooo
FACTOR/ml ADDED
Fig. I. G r o w t h rate of C. briggsae cultures a t v a r i o u s c o n c e n t r a t i o n s of g r o w t h factor a d d e d to chemically defined m e d i u m . ~ - - - - - A , s u p p l e m e n t e d m e d i u m held at 4 °, o v e r n i g h t ; © - - O , s u p p l e m e n t e d m e d i u m frozen o v e r n i g h t a t - - 2 5 ° , a n d t h a w e d i m m e d i a t e l y before inoculation (freeze a c t i v a t e d ) . I n d i v i d u a l cultures c o n t a i n e d 0.25 m l of chemically defined m e d i u m cont a i n i n g g r o w t h factor at t h e c o n c e n t r a t i o n s indicated. C u l t u r e s were i n o c u l a t e d w i t h t h r e e l a r v a e each. T h e n u m b e r of d a y s required for a c o m p l e t e r e p r o d u c t i v e cycle (FI) were d e t e r m i n e d . T h e m a t u r a t i o n rate i n d e x is s h o w n as a f u n c t i o n of c o n c e n t r a t i o n .
obtained in the absence of activation. It can also be seen that above certain concentrations freeze activation does not give the full biological capability of the growth factor. This finding remains to be explained. The time required for complete freeze activation of growth factor was found to be a function of concentration of growth factor in the chemically defined medium (Table I). Activation occurs more rapidly at higher concentrations of protein and complete activation was obtained within 15 min. It is of interest to note that samples frozen in liquid N~ did not activate if thawed and tested immediately. If, however, they were warmed to --25 ° , activation proceeded normally in the times shown for --25 °. Conversely if activated growth factor was frozen in liquid Nz it remained Biochim. Biophys. Acta, 16o (1968) 2o4-216
207
GROWTH FACTOR ACTIVATION TABLE
I
TIME--CONCENTRATION
DEPENDENCE
OF F R E E Z E
ACTIVATION
G r o w t h f a c t o r , a d d e d t o defined media at varying c o n c e n t r a t i o n s , w a s f r o z e n for t h e indicated t i m e s a t - - 2 5 ° . T u b e s w e r e t h a w e d and inoculated by t h e u s u a l a s s a y p r o c e d u r e . R e s u l t s are s h o w n as % maximal activity for t h e c o n c e n t r a t i o n s s h o w n , i.e., percent activation.
Time of freezing (h)
Growth factor (tzg/ml)
Percent activation by freezing 25o
5o
37.5
25
I2.5
O.2 5
IOO
O
O
O
O
0.50
ioo
7o
io
o
o
I.O
I00
IO0
80
50
o
2,o
I0O
I00
IO0
IO0
IO
4.o
IOO
IOO
IOO
IOO
IO0
IOO
IOO
IOO
IOO
IOO
8
9
64.0 C o n t r o l F~ t i m e (days)
4
4½
6½
activated when thawed quickly in a water bath and assayed for biological activity. The reversible nature of activation by freezing is shown in Table II. Aliquots of growth factor in defined medium were activated by the normal freeze-activation procedure and held for varying times at 37 ° . At the appropriate time, these samples were tested without refreezing and after refreezing to determine the residual activation and also the potential activity after complete activation by refreezing. At 37 °, activation (by freezing) begins to decay and by 24 h has completely decayed. Refreezing, TABLE
II
GROWTH FACTOR: FREEZE-ACTIVATION
DECAY--REACTIVATION
S a m p l e s , c o n t a i n i n g 7 5 / z g / m l o f g r o w t h f a c t o r i n chemically defined m e d i u m , w e r e f r o z e n ( -- 25 °) o v e r n i g h t for r o u t i n e a c t i v a t i o n . S a m p l e s w e r e w i t h d r a w n a t a p p r o p r i a t e t i m e s a n d s t o r e d for t h e i n d i c a t e d t i m e s a t 37 ° . A s s a y t u b e s w e r e i n o c u l a t e d w i t h l a r v a e a f t e r o v e r n i g h t s t o r a g e a t 4 ° ( n o t r e f r o z e n ) , or a f t e r o v e r n i g h t h o l d i n g a t - - 2 5 ° (freeze activation repeated). Similar decay o f f r e e z e activation and reactivation by r e f r e e z i n g w e r e a l s o s h o w n a t 3 o°, a n d a t 20 ° ; (see t e x t ) . T h e m a t u r a t i o n t i m e s s h o w n are t h e a v e r a g e o f d u p l i c a t e a s s a y s .
Time at 37 ° after original activation (h) o (control) I 2 3 8 24
Maturation time (days) Not refrozen Refrozen 7.5
ii n.m.*
6.5 6.75 6.75 6 6 6.5 °
* Non-maturing
a t 2 i days.
7 7 8.25
however, restores the full biological activity of the material, thus establishing the reversible nature of the activation process when accomplished by freeze activation. Similar decay of freeze activation and reactivation by refreezing were also demonstrated at 3 °o and at 20 °. Decay of activation was slower and some residual activation remained after 48 h at 3 °0 and after 3 days at 20 °. Complete activation could, in every case, be restored by refreezing. Biochim. Biophys. Acta, 16o (1968) 2 o 4 - 2 1 6
208
F. W. S A Y R E , M. C. F I S H L E R , M. E. J A Y K O
TABLE III EFFECT
OF H E A T I N G
ON G R O W T H F A C T O R A C T I V I T Y
G r o w t h f a c t o r s o l u t i o n s (I m g / m l ) w e r e t r e a t e d as i n d i c a t e d . S u i t a b l e a l i q u o t s w e r e a d d e d t o d e f i n e d m e d i u m t o g i v e 5 ° / ~ g / m l g r o w t h f a c t o r a n d t e s t e d for b i o l o g i c a l a c t i v i t y .
Conditions
4 °, o v e r n i g h t 37 ° , o v e r n i g h t 53 °, 5 m i n 7 o°, 5 m i n IO°°, 5 m i n Autoclaved, t5 min
Mataralion time (days) Unfrozen"
Frozen**
n.m.*** 5-5 5.75 n.m. n.m. n.m.
4-5 5 5 n.m. n.m. n.m.
Unfrozen (not activating conditions). "" A f t e r f r e e z i n g o v e r n i g h t a t - - 2 5 ° ( a c t i v a t i n g c o n d i t i o n s ) . "*" n . m . , n o n - m a t u r i n g a t 21 d a y s . M a t u r a t i o n t i m e s are a v e r a g e of d u p l i c a t e a s s a y s .
Heat activation
The effect of heating on activity of growth factor is shown in Table III. Incubation of growth factor alone at 37 ° overnight produces essentially complete activation ; subsequent freeze activation in defined medium produces only a slight increase in activity under these conditions. At 53 ° for 5 min nearly complete activation of growth factor is observed. Subsequent freeze activation in defined medium brings this to full activity. 7 °° for 5 rain irreversibly destroys all biological activity of the growth factor TABLE IV HEAT ACTIVATION--CONCENTRATION DEPI!NDENCE S a m p l e s o f g r o w t h f a c t o r w e r e h e a t e d a t t h e i n d i c a t e d c o n c e n t r a t i o n s a n d t e m p e r a t u r e s for 2.5 m i n . T u b e s w e r e p r e h e a t e d t o t e m p e r a t u r e b e f o r e a d d i t i o n of g r o w t h f a c t o r s o l u t i o n s . A f t e r h e a t i n g s a m p l e s w e r e c h i l l e d i n a n ice b a t h . A n e q u a l a m o u n t o f d e f i n e d m e d i u m w a s a d d e d a n d s a m p l e s w e r e d i v i d e d i n t o f o u r t u b e s . T w o w e r e t e s t e d a f t e r s t a n d i n g o v e r n i g h t a t 4 ° (nona c t i v a t i n g ) , a n d t h e o t h e r t w o w e r e f r o z e n (for a c t i v a t i o n ) . All s a m p l e s w e r e i n o c u l a t e d a t t h e s a m e t i m e a n d f o l l o w e d b y t h e s t a n d a r d a s s a y p r o c e d u r e . T h e t i m e s for r e p r o d u c t i o n are t h e a v e r a g e of d u p l i c a t e a s s a y s .
Temp.
Maturation time (days) Activation level Test level
4001~g/ml" 20o #g/ml*
zoo #g/ml* 50 #g/ml*
50 I~g/ml * 25 t~g/ml"
Unfrozen
Frozen
Unfrozen Frozen
Unfrozen Frozen
5 °0 51° 520 53 ° 54 ° 55 °
n.m. n.m. 9.5 5.5 5.5 5
4.25 4 4 4.5 4.5 4-5
n.m. n.m. n.m. n.m. 9 8
4.5 4-5 5 4.5 5.5 5.5
n.m. n.m. n.m. n.m. n.m. n.m.
5.5 7 5.5 6 5-5 6
N o h e a t i n g (control)
n.m.
4-5
n.m.
5
n.m.
6
* Growth factor concentration.
Biochim. Biophys. Acta, 16o (1968) 2 o 4 - 2 1 6
209
GROWTH FACTOR ACTIVATION
either with or without freeze activation. The same irreversible destruction of growth factor activity was found after heating at IOO° for 5 rain or after autoclaving. It was thus shown that heating can activate the growth factor or completely destroy all biological activity in an irreversible manner depending on concentration, temperature, and time of exposure. Heat activation was found to be highly dependent upon concentration at various temperatures (Table IV). Under these conditions, partial activation of growth factor by controlled heating can be brought to full activation by subsequent freezing in chemically defined medium. This suggests that the two processes are related. Heat activation at the body temperature of mammals was also found to be highly concentration dependent (Table V). No evidence of heat activation (37 °) was obtained at
TABLE V ACTIVATION BY HEATING (CONCENTRATION DEPENDENCE) Samples of g r o w t h factor, at the indicated concentrations, were held at 37 ° for 24 h (Column i). The heated g r o w t h factor was bioassayed at 75/~g/ml in defined m e d i u m - - w i t h o u t activation (4 ° overnight in defined medium) and after activation b y freezing (--25 ° overnight in defined medium). M a t u r a t i o n times s h o w n are the average of triplicate assays for each point, n.m., non-maturing.
Growth factor concentration
Maturation time (days)
Heated (l~g/ml)
Tested (l~g/ml)
Unfrozen Frozen
3 oo° 15oo 75 ° 45 ° 15° Unheated
75 75 75 75 75 75
6.5 n.m. n.m. n.m. n.m. n.m.
5.5 5.5 5.5 5.5 5.5 5.5
low concentrations of growth factor, even though these were still considerably above physiological concentrations of growth factor. The average physiological concentration of circulating growth factor is about 70/~g/ml. This indicates that physiological activation is probably not accomplished by controlled heating since relatively high concentrations of growth factor are required for activation at physiological temper atures. Mixture with metalic cations
Certain divalent cations have been shown to activate growth factor while other metalic ions are without effect under the conditions tested (Table VI). The relative concentrations of metal to protein are important. Magnesium, calcium, manganese and cadmium showed no activation of growth factor under these conditions, however, freeze activation occurred normally after exposure to these (metal ions). Manganese ion showed a possible slight stimulation of activity after freeze activation. The sulfhydryl-blocking reagents were shown to destroy biological activity of growth factor. Another group of compounds (EDTA, oxidized and reduced glutathione and dithioBiochim. Biophys. Acta, 16o (1968) 2o4-2x6
210
F. W. SAYRE, M. C. FISHLER, M. E. JAYKO
T A B L E VI METAL
ION
ACTIVATION
All i n c u b a t i o n s were for i 8 h a t p r o t e i n c o n c e n t r a t i o n s of i o o o / ~ g / m l a n d a g e n t a t i mM (unless o t h e r w i s e i n d i c a t e d ) a t 4°; b i o a s s a y s were done w i t h a n d w i t h o u t freeze a c t i v a t i o n a t i o o / ~ g p r o t e i n / m l . E s s e n t i a l l y t h e s a m e r e s u l t s were o b t a i n e d a t g r o w t h fa c t or c o n c e n t r a t i o n s of 25 ~ g / m l . The s a m p l e freeze a c t i v a t e d in defined m e d i u m , w i t h o u t a n y a d d i t i o n , w a s a c c e p t e d as t he i oo % s t a n d a r d of a c t i v i t y for t h e s e e x p e r i m e n t s .
Agent added
% Activation Without freeze activation
None MgCI~ CaC12 MnCI 2 CaC12 HgC12 N-Ethylmaleimide EDTA G l u t a t h i o n e (reduced) G l u t a t h i o n e (oxidized) Dithiothreitol CoCI~ Fe(NH4) 2SO~ CuSO 1 NiC12 ZnC12 (o.oI M) ZnC12 (o.ooi M)
o
o o o o o o o o o o 86 82 69 ioo 7o o
Freeze activated*
I oo * ~
Ioo ioo 13 ° ioo o o 9o Ioo ioo I oo Ioo Ioo 72 9o 48 ioo
* After freezing in defined m e d i u m to give n o r m a l a c t i v a t i o n conditions. ** 4,5 d a y g e n e r a t i o n time. (MRI = I / F j x i 0 0 0 = 222.2) IOO~0a c t i v a t i o n .
threitol) showed essentially no effect upon the state of activation of growth factor or upon the ability of growth factor to freeze activate (except for E D T A which showed slight inhibition of activation upon freezing). The next group of metal ions (cobalt, iron, copper, nickel, and zinc) showed some activation of growth factor under the conditions tested. It is of interest to note that cobalt and Fe 2÷ showed incomplete activation of growth factor, yet did not prevent subsequent complete activation by the freeze-activation procedure. Nickel and zinc are of interest in that they showed activation of growth factor, however, a slight depression of the activity (or activation) was observed after freeze activation. ZnC12, at a lower concentration, showed no activation of growth factor but permitted complete activation by freezing. Cu ~+ is of special interest in that it shows different effects, depending on its concentration (Table VII): (I) it shows no effect, (2) can facilitate freeze activation, (3) can activate b y itself, (4) can completely block all activation. At low concentrations, Cu 2+ shows no effect upon the state of activation and shows no interference in the freeze-activation process. At o.I mM, Cu *+ produces a 53 % activation without freeze activation, however, upon freezing, activation becomes complete. At I mM, where Cu 2+ gives an 8o °/o activation, upon freezing only 55 ~/o of the control biological activity was found. At o.oi M, Cu 2+ not only did not activate but prevented the freeze activation. Inhibition of growth factor activity at high concentrations of Cu 2+ is likely Biochim. Biophys. Acla, 16o (1968) 2o4--216
GROWTH FACTOR ACTIVATION TABLE
211
VII
C u 2+ ACTIVATION OF GROWTH FACTOR E x p o s u r e o f p r o t e i n w a s a t IOOO/~g p r o t e i n / m l a t t h e C u 2+ c o n c e n t r a t i o n s s h o w n . B i o a s s a y s w e r e d o n e a t i o o k t g / m l p r o t e i n a n d a t 2 5 / ~ g / m l p r o t e i n . T h e f r e e z e - a c t i v a t e d s a m p l e , w i t h o u t C u 2+, was accepted as the lOO% standard of activity for these experiments.
CuSO 4 conch. % Activation at exposure (raM) Without Freeze freeze activated activation O IO 1 o.I O.Ol o.ooi
O O 80 53 o o
IOO O 55 IOO ioo ioo
due to interaction of copper with the sulthydryl groups of growth factor which are essential for its biological activity 15. Mixture with Ficoll--a synthetic polymer of sucrose distributed by Pharmacia Fine Chemicals--has been shown to activate growth factor. This activation has been discussed by BUECHER,HANSEN AND YARWOOD16. It has since been found that the concentrations of Ficoll required for activation vary with concentrations of growth factor protein and somewhat from preparation to preparation of growth factor. Ficoll is reported to have a molecular weight of approx. 400 ooo. It was designed for densitygradient centrifugation studies and has been presumed to be chemically inert. The only reactive chemical groups of Ficoll presumably are aliphatic hydroxyl groups (about 38 %). We found however that preparations of Ficoll in solution give a crystalline reaction product with 2,4-dinitrophenyihydrazine. This could indicate the presence of either an aldehyde or keto group which theoretically should not be present and may be so only as a result of partial decomposition or impurities of the Ficoll. The parent compound, sucrose, which has the same chemical groups as Ficoll, blocked activation of the growth factor by the freeze-activation process at concentrations comparable to Ficoll on a weight basis. As yet, the mechanism of activation by Ficoll remains unexplained. Activation by mixture with Ficoll does, however, increase chemical reactivity of the sulthydryl groups of growth factor in the same way as other activation procedures 15. Although a great deal is known of the interactions of protein molecules with cationic and anionic molecules 17 very little is known of the interaction of nonionic molecules, such as Ficoll, with proteins. Activation of growth factor by other non-physiological means has been observed under various conditions but has been inconsistent. Low ionic strength and pH of buffers have been observed to activate growth factor under certain conditions and have been discussed by BUECHER,HANSEN AND YARWOOD16. Attempts to study these conditions of activation in detail have shown them to be inconsistent. Likewise, spontaneous activation occasionally occurs upon prolonged storage, but this too is inconsistent, rare and unexplainable on the basis of the known chemistry of growth factor. Biochim. Biophys. Acta, 16o (1968) 2 o 4 - 2 1 6
212
F. W. SAYRE, M. C. FISHLER, M. E. JAYKO
Evidence of structural changes upon activation Several lines of evidence indicate that structural changes occur during the activation of growth factor, irrespective of the activation procedure. Several of the activation procedures show overlapping in the nature of the evidence for conformational changes following activation of the growth factor. Any one of these indications alone would not be sufficient to establish a conformational change related to the activation process. However, when they are all considered together, they make fairly convincing evidence that critical structural changes are responsible for the activation of growth factor. YANKEELOVAND KOSHLAND18 have evaluated the criteria for conformational changes and pointed out the strength of diverse and overlapping evidence for conformational changes. The specific evidence of a structural change in the protein molecule with activation of growth factor is : Differential reactivity of sulJhydryl groups. Changes in the chemical reactivity of the sulfhydryl groups of growth factor have been observed after activation by heating and by mixture with Ficol115. The maximal number of sulfhydryl groups reacting with
0
"
001
o
i
NONACTIVAT
__./ ~
{
003
m
004
o
005
ACTIVATED
•
BY FREEZrNG
X 5o uJ
006 007
I IO
I 20 MINUTES
I 30
I
40
--
008200
~o
,~o
WAVELENGTH, MILLIMICRONS
~o
Fig. 2. Time course of digestion of g r o w t h factor in defined m e d i u m (e~--O) and g r o w t h factor freeze activated in defined m e d i u m (@ @!. Digestion was followed by base u p t a k e with a R a d i o m e t e r t i t r a t o r used as p H star. The p H was adjusted to 7.8o before the addition of t r y p s i n and digestion was allowed to proceed. Values are corrected so t h a t each point is due only to H + liberated by digestion. Samples contained g r o w t h factor, 2Io #g in defined m e d i u m and 20/tg t r y p s i n in a final volume of i .o ml. The samples were identical except t h a t one was activated by freezing overnight at --25 °. Only the first 35 rain are shown, b u t total base u p t a k e was the same for the two samples after 24 h. Similar results were obtained with Ficoll activated g r o w t h factor. Fig. 3. 0 - - 0 , optical r o t a t o r y dispersion of g r o w t h factor (non-activated); Q - - © , same of g r o w t h factor activated with Ficoll at 5 % (corrected for Ficoll rotation). The samples were growth factor at i mg/ml, in o.i M p o t a s s i u m p h o s p h a t e buffer; and g r o w t h factor at i m g / m l activated in o.I M p o t a s s i u m p h o s p h a t e buffer containing 5 % Ficoll. The p H of b o t h buffer solutions was 7.0. The curves are corrected for buffer and for buffer plus 5 % Ficoll so t h a t the values s h o w n are the r o t a t i o n s observed for the protein solutions a]one, non-activated, and after activation b y m i x t u r e with Ficoll. Activation by Ficoll and non-activation of the control were confirmed b y bioassay of aliquots of each sample. These determinations were made on a Cary-6o optical r o t a t o r y dispersion a p p a r a t u s b y Dr. M. JAYKOat the University of California.
Biochim. Biophys. Acta, 15 ° (1968) 2o4-216
GROWTH FACTOR ACTIVATION
213
p-chloromercuribenzoate was found to be the same for activated and non-activated growth factor while their spatial arrangement appears to be different as evidenced by their different rates of chemical reaction. Susceptibility to trypsin digestion. The time course of digestion of non-activated growth factor is a smooth one, whereas, digestion of growth factor (freeze activated) occurs in discrete steps (Fig. 2). The total number of bonds split by trypsin after 24 h digestion is the same for activated and for non-activated growth control factor. Similar results were obtained with growth factor activated by Cu 2+ and by mixture with Ficoll. ]'he step-wise digestion of activated growth factor by trypsin indicates activated growth factor to be a more highly ordered structure than non-activated growth factor. The immediate splitting of one peptide bond per 21 ooo g of growth factor protein indicates that one lysine (or arginine) group is more susceptible to tryptic digestion in activated growth factor than in non-activated growth factor, probably by virtue of its greater accessibility to trypsin. The same findings after activation of growth factor by three different procedures suggest similar structural changes in this region of the molecule. Titratable groups. Non-activated growth factor shows 14 groups per 21 ooo g of protein titratable at the pK of e-aminolysine (Table VIII). Growth factor activated by freeze activation shows only 5 of these groups. Amino acid analysis showed 14 lysine TABLE VIII T I T R A T A B L E G R O U P S OF G R O W T H F A C T O R
T i t r a t i o n s w e r e done w i t h a R a d i o m e t e r p H s t a t in defined m e d i u m . All v a l u e s are c orre c t e d for defined m e d i u m , so are n e t for protein.
p H range
7 . 0 - 8.0 8 . 0 - 9.0 9.0 io,o io.o-ii.o I I.O--I2,0
Titrated groups (equiv/2i ooo g protein) Non-activated
d c!ivated
4.4 I2.2 25. 9 13. 9 I6. 3
0.8 0.8 2. 5 4.9 3.9
groups per 21 ooo molecular weight. This would suggest all e-aminolysine groups are available in the non-activated condition and 9 are folded in after activation 19. These findings also provide good substantiation of the amino acid composition and of the 21 ooo molecular weight basic unit. Optical rotatory dispersion. The optical rotatory dispersion of growth factor and of Ficoll activated growth factor are shown in Fig. 3. Non-activated growth factor shows minimum Cotton Effect at 238 m/~ which shifts to approx. 235 m# after activation with Ficoll z0. There is also a pronounced decrease in levorotation, indicating a more ordered structure zl. A crossing of the zero axis of rotation by the activated growth factor (corrected for Ficoll rotation) furthermore indicates a conformational change within the protein. Since the optical rotatory dispersion curves were determined only after activation with Ficoll, the optical rotatory dispersion studies can only be used Biochim. Biophys. Acta, i 6 o (1968) 2o4-216
214
F.w.
SAYRE, M. C. FISHLER, M. E. JAYKO
as confirmatory evidence of the other indications of conformational changes which occur with activation. Viscosity changes with activation. In a typical experiment, a solution of growth factor at 2oo #g/ml in chemically defined medium was found to have an efflux time of 315.14 sec from the capillary of a Ubbelohde viscometer, corresponding to a value of o.84826 centistokes. After activation by freezing overnight, another aliquot of this same solution was found to have an efflux time of 323.o 9 sec, corresponding to o.86966 centistokes. The first three readings of the efflux time of activated growth factor were in good agreement, but the effiux time of the fourth reading was found to be 315.57 sec or essentially the same as the non-activated growth factor. This reading remained constant. Control bioassays indicated that the growth factor had been activated when the original viscometric measurements were made and that after passage through the viscometer three times, the material was no longer activated, but could be reactivated upon refreezing. Thus, the shearing action of passing activated growth factor through the capillary tube of the viscometer resulted in its biological deactivation as well as a return of the efflux time to that of nonactivated growth factor. Interaction with physiological inhibitor. Physiological inhibitor of growth factor activity has recently been isolated from human serum and shown to interact with growth factor; this interaction was shown by ultracentrifugal analysis and by measured changes in the chemical reactivity of the sulthydryl groups of growth factor. The inhibitor of growth factor activity is a new protein which reacts quantitatively with growth factor and suppresses its biological activity in the nematode maturation assay ~. The suppressive effect of natural inhibitor on growth factor activity can be overridden by natural activator (in higher concentrations) or by Ficoll (a powerful non-physiological activator).
DISCUSSION
Similar quantitative changes in the biological activity of growth factor have been observed in the nematode maturation assay following activation by widely varying procedures. There has been evidence of a conformational change in the protein following each of the activation procedures 2=. For technical reasons it has been impractical to apply all criteria cited for conformational changes to all activation procedures. It has, however, been possible to detect sufficient overlap in these criteria to provide evidence of similar structural changes following different activation procedures. For example: (I) Similar changes in the susceptibility of growth factor to tryptic digestion were found after activation by freezing in defined medium, controlled heating, mixture with Ficoll and mixture with Cu 2+. (2) Similar changes in the chemical reactivity of the sulthydryl groups of growth factor were found after activation by controlled heating or mixture with Ficoll; sulihydryl groups are of special interest as they have been shown to be essential to biological activity. (3) Similar changes in the titratable groups of growth factor have been observed following activation by freezing, mixture with Ficoll, and mixture with Cu =+; this shows different degrees of exposure of specific chemical groups of growth factor in the activated and non-activated condition. (4) Changes in the optical rotatory dispersion have been observed following activation with Ficoll. This overlapping evidence of conformational Biochitn. Biophvs. Acta, 16o (I968) 2o4-216
GROWTH FACTOR ACTIVATION
215
changes following activation by different procedures indicates that critical structural changes must occur during activation by any of these procedures. Reversibility of most of the activation procedures described further substantiates the idea of a reversible conformational change within the protein molecule and its relationship to the activation mechanism. The finding that partial activation by one procedure can be brought to full activation by a second and different activating procedure indicates that similarities exist in the different activation procedures. All of these evidences considered together suggest that critical conformational or structural changes occur during activation which correlate with changes in the observed biological activity of growth factor. The various methods of activation described above have resulted in increased or modified biological activity in other systems such as cell culture in vitro, bacterial growth, and retardation of growth of transplanted tumors. Activation by mixture with Ficoll and by freezing in chemically defined medium (or nucleotides) has been observed in all biological systems studied thus far, and has been found to alter significantly the biological activity of growth factor in each system. Activation by controlled heating has been demonstrated in all biological systems studied except in the growth of transplantable tumors (where it has not been tried). Cu 2+ and other metal ion activation have been studied only with the nematode maturation assay. Activation of a number of enzymes by similar means has been observed for some time. One of the earliest recognitions of this phenomenon can be credited to HOFSTEE23 and came from his studies on urease activity. Reversible activation of urease was described by controlled heating and by freezing under certain conditions. More recent work indicates that these activations are the result of conformational changes within the protein molecule. Recent specific examples of conformational changes accompanying altered biological activity (i.e., activation and deactivation) have been clearly shown for: (I) phosphoglucomutase24; (2) alkaline phosphatase from Escherichia c01i25; and (3) dihydrofolate reductase from L-I2IO cells26. In all of these examples, limited conformational changes lead to activation of the enzyme--usually reversible. More extensive conformational changes, often by the same agents, result in irreversible inactivation of biological activity. These findings parallel and overlap in many ways the findings with growth factor and suggest that such regulatory mechanisms are far more common for regulation of biological processes than had been heretofore recognized. The discovery of the physiological activator and inhibitor as well as growth factor in the sera of man and other mammals is significant from several points of view. First, it provides a physiological control mechanism for the above-described conformational changes in the growth factor. Second, it gives further meaning to the work described herein on nonphysiological activation procedures. Third, it provides a start in the understanding of a newly recognized homeostatic mechanism possibly concerned with cellular growth in mammals.
ACKNOWLEDGMENT
This investigation was supported in part by Grant AM-o6oo4 from the National Institutes of Health, U.S. Public Health Service. Biochim. Biophys. Acta, 16o (1968) 2o4-216
216
F. W. SAYRE, M. C. FISHLER, M. E. JAYKO
REFERENCES i F.W. SAYRE, E. HANSEN AND E. A. YARWOOD, Exptl. Parasitol., 13 (1963) 98. 2 F. W. SAYRE, R. T. LEE, R. P. SANDMAN AND G. PEREZ-MENDEZ, Arch. Bioehem. Biophys., 118 (1967) 58 . 3 F. W. SAYRE AND M. C. FISHLER, s u b m i t t e d for publication. 4 T. J. STARR, M. POLLARD, Y. TANAMI AND R. MOORE, Texas Rep. Biol. Med., 18 (196o) .5Ol. 5 F. W. SAYRE, E. NANSEN, T. J. STARR AND E. A. YARWOOD, Nature, 19o (I961) 1116. 6 F. W. SAYRE, E. I-{ANSEN, P. BARNARD AND E. A. YAR~VOOD, Federation Proc., 21 (1962) 238. 7 P. H. SILVERMAN, E. L. HANSEN, E. BUECHER, J. Parisitol., 51 (1965) 46. 8 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND m. J. RANDALL, jr. Biol. Chem., I93 (1951) 265 . 9 H. ~(-. SCHACHMAN, Ultracentrifugation in Biochemistry, Academic Press, New York, 1959, p. 63 and 181. lO A. M. KRAGH, A Laboratory Manual of Analytical Methods of Protein Chemistry, Vol. 3, Perg a m o n Press, L o n d o n 1961, p. 173. l 1 E. HANSEN, F. ~V. SAYRE AND E. A. YARWOOD, Experzentia, 17 (1961) 32. 12 P. D. BOYER, J. Am. Chem. Soc., 76 (1954) 4331. 13 N. M. ALEXANDER, Anal. Chem., 3 ° (1958) 1292. 14 C. F. JACOBSEN, J. LEONIS, K. LINDERSTROM-LANC AND M. OTTESEN, Methods of Biochemical Analysis, Vol. 4, Interscience, New York, 1957, p. i. 15 F. W. SAYRE, M. C. FISHLER, G. HIJMPHREYS .aND M. E. JAYKO, Biochim. Biophys. Acta, 16o (I968) 63. 16 E. J. BUECHER, E. HANSEN .aND E. A. YARWOOD, Proc. Soc. Exptl. Biol. Med., I2I (1966) 39o. I 7 P. BERNFELD, B. J. BERKELEY AND R. E. BIEBER, Arch. Biochem. Biophys., II1 (1965) 31. 18 J. A. YANKEELOV AND D. E. KOSHLAND, JR., J. Biol. Chem., 240 (1965) 1593. I9 C. TANFORD, Physical Chemistry of Macromolecules, J o h n \Viley and Sons, New York, I96t, p. 564 . 20 C. TANFORD, Physical Chemistry, of l~Iacromolecsles, J o h n Wiley and Sons, New York, 196I, p. 119. 21 \V. F. HARRINGTON, R. JOSEPHS AND D. M. SEGAL, Ann. Rev. Biochem., 35 (1966) 599. 22 S. N. TIMASHEFF AND M. J. GORBUNOFF, Ann. Rev. Biochem., 36 (1967) 13. 23 13. J. HOFSTEE, J. Gen. Physiol., 32 (1948) 339. 24 V. BOCCHINI, M. R. ALIOTO AND V. A. NAJJAR, Biochemistry, 6 (1967) 3242. 25 J. A. REYNOLDS AND M. J. SCHLESINGER, Biochemistry, 6 (1967) 3552. 26 P. REYS AND F. M. HUENNEKENS, Biochemistry, 6 (I967) 3519.
Biochim. Biophys. Acta, i6o (I968) 2o4-216