c 3T3 cells

74

Btochimica et Biophysica Acta, 1053 (1990) 74-80 Elsevier

BBAMCR 12714

Stimulatory effect of serum albumin on the proliferation of serum-free SV40-transformed Balb/c 3T3 cells R o b e r t a B e d o t t i 1, A n g e l o F. B o r g h e t t i 2 a n d R o b e r t o F a v i l l a l J Section of Biophysics and Molecular Biology. Department of Physics and ; Institute of General Pathology, University of Parma. Parma (Italy) (Received 9 February 1990)

Key words: Serum albumin; Cell proliferation; Serum-free media; Mitogenicity; (SV3T3 cells)

Commercial serum albumins have been found to be able to stimulate the proliferation of B a l b / c 3T3 calls transformed by SV40, but not that of the normal counterpart. The effect is most pronounced with cristalline samples of albumin depleted of both globulin and fatty acid components, and depends on conditions used for the attachment and on seeding density. Physical and chemical treatments aimed to remove tightly bound impurities do not abolish the activity of fatty acid free serum albumin, thus supporting the idea that albumin per se is mitogenic towards these cells.

Introduction It is now a well established fact that transformed cells require little or no serum supply to support their growth in culture [1]. This behaviour has been related to the acquisition of an autocrine mechanism, according to which transformed cells can secrete growth factors or growth factor-like molecules recognized by specific receptors on their cellular surface [2]. SV40 transformed 3T3 cells are able to grow in the complete absence of serum, although their generation time becomes substantially longer and their saturation density lower [3,4]. Later is has been reported that SV40-transformed Balb/c as well as Swiss 3T3 cells show a growth rate and a saturation density similar to that observed in 10% FCS, when supplemented with a defined medium composed of insulin, transferrin, fatty acid-free BSA and linoleic acid [5]. In that paper the authors state that fatty acid-free BSA has no growthpromoting activity by itself but it is required to prevent the cytotoxic effects of free fatty acids. In this paper we

Abbreviations: SV3T3, 3T3 cells transformed by SV40 virus; FCS, foetal calf serum; DMEM, Dulbecco's modified minimal essential medium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RSA, rat serum albumin; HoSA, horse serum albumin; HuSA, human serum albumin; No. 5-, Cohn's fraction 5 (albumin); GF-, globulin-free (albumin); FAF-, fatty acid-free (albumin); 4PDS, 4,4'dithiopyridine; TGF-fl, transforming growth factor ft. Correspondence: R. Favilla, Sezione di Biofisica e Biologia Molecolare, Dipartimento di Fisica, Universit,q degli Studi di Parma, Viale delle Scienze, 43100 Parma, Italia.

show that SV40-transformed Balb/c 3T3 cells can grow in a medium supplemented only with fatty acid-free serum albumin. A similar effect has previously been described in the case of activated lymphocytes [6,7]. A growth-promoting effect is also observed with other commercial preparations of BSA containing fatty acids, though in a less pronounced way, depending upon their degree of purification. In contrast, the untransformed cellular counterpart (Balb/c 3T3 cells) is not able to grow under the same conditions. Materials and Methods

Cells Normal and SV40-transformed Balb/c 3T3 cells were obtained from the American Type Cell Culture collection. SV3T3 stock cell cultures were transferred once or twice weekly and maintained in DMEM (4.5 g/l glucose) supplemented with 5% FCS and containing penicillin (100 IU/ml), streptomycin (0.1 mg/ml) and sodium bicarbonate (3.7 g/l) in 81 cm2 plastic Petri dishes (Costar) at 37°C in a humidified 5% CO2 air atmosphere. Cell harvesting Exponentially growing cells were washed with PBS, incubated for a few minutes with 0.05% trypsin in PBS, then 0.05% soybean trypsin inhibitor in PBS was added. The cell suspension was centrifuged at 1500 rpm for 5 rain and the pellet was resuspended in DMEM plus 0.5% FCS. For experiments, cells were seeded into 4-cm2 dishes and supplied with the appropriate medium.

0167-4889/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

75 After 24 h the medium was replaced with fresh, and the appropriate components were added. Cell number was determined with a model ZM Coulter counter. In the figures the mean values of duplicate dishes are reported. DMEM and FCS were from Gibco. Linoleic acid as well as all proteins used, (trypsin, soybean trypsin inhibitor, bovine insulin, human transferrin, bovine, human, horse and rat albumins, bovine fetuin, lysozyme, a-chimotrypsin, globin and heparin and 4,4'-dithiodipyridine) were from Sigma.

4-PDS in the same solvent. The reaction was monitored to completion at 320 nm, then the solution was dialysed and lyophilised. NaCI and GuCL BSA samples were dissolved in either 2 M NaC1 or 6 M GuCI at pH 7, then dialysed and lyophilised. The mitogenic activity of each of these samples was tested 48 h after addition to cells. Results

Physico-chemical treatments of Cohn's fraction No. 5-BSA and fatty acid-free BSA Trypsin. 100 ml of 5% BSA in 0.1 M NaC1 at pH 7.4 were added of 10 mg trypsin (1 h at 37°C) then of 10 mg trypsin inhibitor (16 h at 4°C) followed by extensive dialysis against water (12-14 kDa cutoff dialysis membrane) and lyophilisation. Detoxi-gel. 40 mg BSA in 10 mM phosphate plus 0.1 M NaC1 at pH 7 were passed trice through a 10 ml column filled with 3.5 ml detoxi-gel (Pierce Chemical Co.), then dialysed and lyophilised, as described above. HPLC-gelfiltration. 50 mg BSA in 1 ml of either 0.1 of 1 M acetic acid plus 0.1 M NaCI were chromatographed through two serially mounted Waters columns (PP125 and PP60, 0.8 × 30 cm) each, using a model 510 HPLC Waters system. About 40 1-ml fractions were collected, dialysed and lyophilised. 4-PDS. 40 mg BSA in 40 ml 50 mM phosphate plus 0.15 M NaCI at pH 7.4 were added of 1 ml of 2 mM

Growth of cells in serum-free medium The effect of seeding density on the growth of SV3T3 cells was investigated as a function of added FCS or BSA concentration. After 24 h of incubation with different percentage of FCS or BSA, the cells were washed twice with DMEM and then incubated with DMEM alone. Proliferation was followed at different times for a maximum of 4 days from deprivation (Fig. 1). In contrast to FCS, BSA alone in the seeding medium does not induce proliferation even at a high concentration (4 mg/ml), independently of the seeding density.

Effect of BSA on cell proliferation In another set of experiments, both SV3T3 and 3T3 cells were inoculated at a density of 2.0. 104/cm 2 in 0.5% FCS. Cells were serum deprived after 24 h and added of variable amounts of BSA (and of FCS as a control). Three different preparations of BSA (Cohn's

600

2°t

A

50O

B

/jo

f,1

0400

o 15

)4

~E 300 u

u 10

_~ 2oc u

5

%

100

0 24

48

6oo[ c

72 Time (h)

96

1--20

i

0

24

m

48

-

72

--

96

m

I~0

Ttme ( h )

o ~ o .

% ×

3oo

0

7

24

48

\o

72 Time (h)

96

120

Fig. 1. Growth of SV3T3 cell in serum-free medium. Cells were seeded at three different densities (in cells/cm2); A, 2000; B. 10000: C. 50000, and with different FCS levels: ~, 107o; O, 7.570; I , 5%: A 2.570; v, 1.2570; m, 0.62570; or with 4 m g / m l FAF-BSA, zx. 24 h later culture media were replaced with D M E M alone and cells were counted during the following 4 days.

76 FCS

,,

0 250~- A

5

2oo'

~

sv4oao,0,~3r3

FCS */.

*/.

10

"

0

'o

×

X

50-

//A I

v

iZ ~ o ~ O O~ ,

1

~

c

10

I ao,~3~3

~

'o

v.

5

15

20

r --------~'P-----------~

9o

6o

-

-'-

,

2 BSA ( m g / m l )

3

4

2

4 BSA ( r r ~ j / m l )

Fig. 2. Effect of BSA on SV3T3 and 3T3 cell proliferation. (A) SV3T3 and (B) 3T3 cells were both seeded at a density of 20000/cm 2 in DMEM containing 0.5% FCS. 24 h later both culture media were replaced with fresh DMEM containing either FCS (e) or different BSA species; FAF-BSA (zx); GF-BSA (v) or No. 5-BSA (O). Cells were then counted after 48 h.

fraction No. 5 (No. 5-), globulin-free and fatty acid-free) were used, ranging from 0.25 to 4 m g / m l with SV3T3 and from 1 to 8 m g / m l with 3T3 cells (4 m g / m l of BSA in equivalent to the amount present in 10% FCS). The proliferative index was then measured 48 h later

I

/

2o~ o 15-

(Fig. 2). The transformed cells are able to grow even in the presence of BSA alone, reaching a plateau value at about 2 mg/ml. This value depends on the different type of commercial BSA preparation used, the fatty acid-free BSA resulting as the most active one. In

/ y"

r~

250- B 2ooL

× 150r

v 10 L ¥

5-

×

~Ev =in

IOOF

u

I

0

TLme (h)

o

u \

z4

,11

m'

2

48

72

96

Time (h)

500L

4ool-

Q 400~

3oi

F 1001..,L O'r 0

I001. 24

48 Time (h)

72

96

OC)

24

4,8 Ttme (h)

f3 72

N 96

Fig. 3. Dependence of BSA response on SV3T3 seeding density. Cells were seeded in 0.5% FCS at the following four densities: (A) 2000; (B) 5000; (C) 20000; and (D) 50000 cells/cm 2. After 24 h, culture media were replaced with fresh DMEM, either alone (o), with 5% FCS (O), with 2 m g / m l FAF-BSA (A), GF-BSA (v), or No. 5-BSA (O). Cells were counted during the following 3 days.

77

1

contrast with this case, BSA does not induce any proliferation of 3T3 cells, even at the highest concentration, keeping the inital n u m b e r of cells [8,9].

Dependence of BSA response on seeding density The effect of different BSA types on SV3T3 cell growth is dependent on the density at which cells are seeded. The effect was followed for 3 days, using cells seeded in 0.5% F C S for 24 h (Fig. 3). T h e critical density above which cells are able to grow depends u p o n the BSA used: with F A F - B S A this value is about 5 0 0 0 / c m 2 with G F - B S A about 20000, whereas with No. 5-BSA above 50000.

Effect of preconditioning treatments and daily medium change on SV3T3 cell growth in the presence of BSA Prolonged overnight washing in D M E M before addition of fresh m e d i u m as well as daily m e d i u m changes in cell cultured in the serum-free, serum-containing or No. 5-, G F - and F A F - B S A - c o n t a i n i n g media have investigated. T w o different seeding densities were used: 15000 and 45 0 0 0 / c m 2. As shown in Fig. 4, some decrease in the mitogenic index of cultured cells is observed in all cases investigated.

Effect of insulin, transferrin and linoleic acid on SV3T3 cells Cells were seeded at a density of 20 0 0 0 / c m 2 in 0.5% FCS containing D M E M . 24 h later, cells were depleted of serum and supplemented with a synthetic m e d i u m made up with different combinations of insulin (2 Ixg/ml), transferrin (5 ~ g / m l ) , and either No. 5-BSA,

500

A ,~

-~ 2 0 0 -

,oo

}

;

S

~, 250 I 0 × 200[

%.o

+

-

÷ 4-

I m ÷

I

o/r"l[q

Treatments

i

Fig. 5. Growth of SV3T3 cells in a synthetic medium. 20,000 cells/cm2 were seeded in 0.5% FCS. After 24 h cells were counted for attachment; then culture media were replaced with fresh DMEM alone or DMEM containing 5% FCS, 2 ~g/ml insulin (1), 5 p.g/ml transferrin (T), insulin plus transferrin (I + T), 2 mg/ml No. 5-BSA, No. 5-BSA plus insulin (+ I), No. 5-BSA plus transferrin (+ T), No. 5-BSA plus insulin and transferrin (+ I +T), 2 mg/ml GF-BSA, GF-BSA plus insulin (+ I), GF-BSA plus transferrin (+T), GF-BSA plus insulin and transferrin (+ I + T), 2 mg/ml FAF-BSA, FAF-BSA plus insulin (+I), FAF-BSA plus transferrin (+T), FAF-BSA plus insulin and transferrin ( + I +T). Cells were counted 48 h later.

G F - B S A or F A F - B S A at 2 m g / m l each. Cells were counted 48 h later. A synergistic effect by insulin and transferrin was obtained with all BSA-species, though mostly p r o n o u n c e d with No. 5-BSA (Fig. 5). The presence of linoleic acid, either alone or with F A F - B S A (in the presence of insulin a n d / o r transferrin) induces cell death already at a concentration as low as 0 . 5 / ~ g / m l (range investigated 0 . 5 - 5 ~ g / m l ; data not shown). This is somewhat surprising, considering that this c o m p o n e n t was previously reported to be necessary for the growth of SV3T3 cells in defined medium [5].

Effect of physio-chemical treatments on BSA activity

B

%~ 4001

÷

";

z

Treatments Fig. 4. Effect of preconditioning treatments and daily medium change on SV3T3 cell growth in the presence of BSA. Cells seeded in 0.5% FCS were treated as follows: conditioned in BSA-containing DMEM after a short wash, conditioned in BSA-containing DMEM after a short wash, conditioned in BSA-containing DMEM after extensive wash, conditioned in BSA-containing DMEM with dialy change of medium. Controls were added of 5% FCS and DMEM alone, respectively after the corresponding preconditioning period. Seeding density: A, 15,000 cells/cm2; B, 45,000 cells/cm2. Cells were counted 72 hours (A) and 48 hours (B) after washing.

In order to remove trace impurities from commercial preparations of BSA, both No. 5- and F A F - B S A were separately submitted to several physical or chemical treatments, described above, and the results are shown in Fig. 6. I n c u b a t i o n with tripsin under mild conditions, appropriate to eliminate an insulin-like activity associated with BSA [10], does not affect the activity of either BSA. C h r o m a t o g r a p h y through detoxi-gel, an endotoxinremoving gel, according to Pierce Chemical Co. [11], or fractionation by H P L C gel filtration under saline acidic conditions does not affect the activity of F A F - B S A but enhances that of No. 5-BSA. Also high-salt (2 M NaCi) or reversible denaturing (6 M GuCI) conditions do not substantially affect the activity of subsequently dialysed F A F - B S A (data not shown). In contrast, reaction with 4-PDS, a thiol-reactive reagent, used to modify the single free thiol group

78

6°°IA--

60C

500F

50C m

4ooL

'o ~ c )<

ooL

30(3

i

2~ 200

3 ~OOL

100 I

OO

24

48 T~me (h)

72

%

g~

2,4

48 Time (h)

96

72

Fig. 6. Effect of physico-chemical treatments of different BSA preparations on the corresponding mitogenic activity. 20000 cells/cm: were seeded in 0.5% FCS. After 24 h, cells were counted for attachment and media were replaced with: A, fresh D M E M alone (©), 5% FCS (O), 2 m g / m l FAF-BSA (A), 2 m g / m l trypsin-treated FAF-BSA (&), 2 m g / m l HPLC-treated FAF-BSA ( I ) , 2 m g / m l Detoxi-gel-treated FAF-BSA (O), and 2 m g / m l FAF-BSA 4PDS-treated FAF-BSA (v); B, same treatments on No. 5-BSA (El). Cells counted during the following three days.

present on BSA [12,13], reduces the mitogenic activity of both BSAs. Growth of SV3T3 cells with other serum albumins

The effect of different mammalian albumins on SV3T3 growth is shown in Fig. 7. In particular, the FAF-serum albumin from rat, horse and man have an effect similar to that exhibited by FAF-BSA, whereas the corresponding No. 5-serum albumins, though slightly more active than No. 5-BSA, are all considerably less mitogenic than FAF-serum albumins. The effects of several other bovine proteins, namely fetuin, heparin, lysozyme, a-chymotrypsin, globin and lactalbumin, as well as those of monomeric and dimeric No. 5-BSA species on cell growth are shown in Fig. 8. In the same figure, the effect of 5% FCS, FAF-BSA and D M E M is also shown for comparison. Among these proteins, only

fetuin, lactalbumin and monomeric and dimeric BSA species show an appreciable effect. Discussion SV3T3 cells are apparently able to sustain their growth even in the absence of serum, but only when they are seeded above a critical density (about 1104/cm 2) and the presence of FCS (approx. 1%), as shown in Fig. 1. In contrast, no autostimulation of SV3T3 cells was observed when BSA alone was present during the preconditioning (attachment) period, even at high seeding dertsity. These results indicate that SV3T3 cells can activate an autocrine mechanism, but only when exposed in advance to the influence of serum factors. SV3T3 cells could no be seeded with BSA alone

I 400~

u.

300

i t~

o 3o0-

u.

i

~

~

2oc

e

200r ,o

m

2_

a~

E

~

_~

~E ; 100

c

100 a

OL

fi

Ot Treotments

Fig. 7. Effect of various albumins from different sources on the growth of SV3T3 cells. 20000 cells/cm 2 were seeded in 0.5% FCS. After 24 h, cells were counted for attachment and media were replaced with D-MEM alone or DMEM containing 5% FCS, No. 5-BSA, No. 5 RSA, No. 5 HoSA, No. 5 HuSA, FAF-BSA, FAF RSA, FAF HoSA and FAF HuSA. Cells were counted 48 h later.

r - ~ r--n Treclt ments

Fig. 8. Effect of several bovine proteins on the growth of SV3T3 cells (all proteins tested were at 2 mg/ml). 20000 cells/cm 2 were seeded in 0.5% FCS. After 24 h cells were counted for attachment and media replaced with DMEM alone or containing 5% FCS, FAF-BSA, fetuin, heparin, lysozyme, a-chimotrypsin, globin, No. 5-BSA monomer, No. 5-BSA dimer, lactalbumin. Cells were counted 48 h later.

79 because they were not able to attach properly in complete absence of FCS [14]. Therefore, in order to get rid of serum factors, cells were left extensively in D M E M alone (overnight washing) before addition of fresh medium containing BSA. Following this treatment, some decrease in cell number was oberved (Fig. 4), which was however only slightly greater than that in the control (FCS). This effect may be attributed to the overnight cell loss due to the stringent conditions used. A similar decrease was also observed when the medium was changed dialy in both treated and controls samples. In this case, the observed decrease could be related to the periodic zeroing of autocrines level in the medium. In both case, however the mitogenic effect of BSA was still substantial. The proliferative index of SV3T3 cells, seeded at 0.5% FCS, was found to be a function of both the concentration and the kind of commercial preparation of BSA added thereafter. The observed efficieny decreased according to the following order: FAF-BSA > GF-BSA > No. 5-BSA (Fig. 2), suggesting a progressive loss of some inhibitor. The maximal effect was reached with FAF-BSA at about 2 m g / m l , a level equivalent to that of BSA in 5% FCS. The autostimulatory effect observed in Fig. 1 with a seeding density of 2 . 1 0 4 was not observed in this case because the level of FCS in the seeding medium was too low (0.5%). In contrast, 3T3 cells were not stimulatd to divide but were still able to survive above 4 m g / m l of BSA, the effect being again somewhat dependent on the degree of purification of BSA. This may be used to keep normal 3T3 cell cultures steady even if not at confluence. When SV3T3 cells, seeded in the presence of 0.5% FCS showed no autocrine mechanism during the conditionment period, independently of the seeding density, but were stimulated to proliferate by addition of BSA, the effect being mostly pronounced with FAF-BSA. The presence of FAF-BSA lowered the critical seeding density necessary for stimulation of proliferation (Fig. 3AD). The fall in cell number observed in Fig. 1A after some days of deprivation can be rationalized assuming that nutrients are used up before cells could reach a density sufficient for their self-sustainment. To test more rigorously the role played by No. 5-BSA and FAF-BSA were subjected to a series of physical a n d / o r chemical treatments in order to see if the mitogenie activity was associated with the BSA molecule itself or some minor impurity. Only treatment with 4-PDS decreased the activity of both BSA samples, thus suggesting a role of the single cysteine residue present on BSA. An increase of activity was observed either after fractionation of No. 5-BSA through HPLC or after its elution from a detoxy-gel column, suggesting the removal of some trace inibitor. Fractionation through HPLC of FAF-BSA under acidic conditions (1

M acetic acid) as well as its treatment with 2 M NaCI or 6 M GuCl, did not appreciably affect the mitogenic activity (data not shown). The overall result is consistent with the idea that the albumin molecule itself is responsible of the observed mitogenicity. In order to assess whether the mitogenic effect elicited by BSA was peculiar to the bovine protein or not, other serum albumins from three different sources (rat, horse, human) were investigated. The general results here obtained agree well with those already mentioned with the bovine albumin, namely also these albumins resulted mitogenic, the efficency decreasing in the order: FAF > G F > No. 5 (Fig. 6). A series of unrelated bovine proteins were also assayed in order to compare their effect with that of BSA. Some of these proteins exhibited mitogenic activity towards SV3T3 cells, the most effective one being fetuin. This fact, however, is not too surprising, considering the role played by fetuin in the attachment of cells to their substrate [15]. As far as the role of albumin is concerned, some mechanisms can be suggested to explain its observed mitogenic activity: (i) Albumin can act as a true growth factor, because SV3T3 cells might have specific receptors. Evidence for the existence of albumin receptor sites was reported for isolated hepatocytes and adipocytes [16]. (ii) Albumin could exploit its effects intracellularly once it has entered the cell by pinocytosis whether or not albumin-specific receptors are present. (iii) Albumin might prevent the loss of autocrine growth factors secreted by the cells cultured in the absence of serum; actually a similar mechanism by albumin towards immunoreactive insulin was shown in cultured rat liver cells [17]; (iv) Albumin might mediate the transport of growth factors or be a carrier of low-molecular-weight mitogens, as observed with crude BSA preparations [10,18]. The series of more or less drastic treatments of FAFBSA performed by us in all cases but one (4-PDS) resulted in no appreciable decrease of albumin activity: this is a good evidence in favour of the fact that FAF-BSA is intrinsically mitogenic towards SV3T3 cells. This effect may be related with the fact that fibroblasts have been characterized as the major sites of albumin catabolism [19]. Finally, it is worth mentioning that no growth-stimulatory activity nor soft agar colony formation was detected with NRK-49F clone cells, in the presence of high doses of FAF-BSA, thus indicating that no appreciable transforming activity like that of TGF-fl, appears to be associated with FAF-BSA.

Acknowledgements This work was supported by grants from C.N.R.. Progetto Finalizzato Oncologia, N. 87.01197.44 and Ministero Pubblica lstruzione, 40% and 60%.

80

References 1 Smith, H.S., Sher, C.D. and Todaro, G.J. (1971) Virology 44, 370-75. 2 Sporn, M.B. and Todaro, G.J. (1980) N. Engl. J. Med. 303, 878-880. 3 Bush, H. and Shodell, M. (1978) Exp. Cell Res. 114, 27-30. 4 Young, D.V., Cox, F.W., 111, Chipman, S. and Hartman, S.C. (1979) Exp. Cell Res. 118, 410-414. 5 Rockwell, G.A., Sato, G.H. and McClure, D.B. (1980) J. Cell. Physiol. 103, 323-331. 6 Spieker-Polet, H. and Polet, H. (1976) J. Biol. Chem. 251,987-992. 7 Polet, H. and Spieker-Polet, H. (1976) J. lmmunol. 117, 1275-81. 8 Holley, R.H. and Kiernan, J.A. (1974) Proc. Natl. Acad. Sci. (USA) 71, 2908-11. 9 Pledger, W.J., Stiles, C.D., Antoniades, H.N. and Scher, C.D. (1977) Proc. Natl. Acad. Sci. USA 74, 4481-85.

10 Jordan, J.E. and Kono. T. (1980) Anal. Biochem. 104, 192-95. 11 PIERCE Handbook and General Catalog (1989) pg. 41. 12 Grassetti, D.R. and Murray, J.F., Jr. (1967) Arch. Biochem. Biophys. 119, 41-49. 13 Regen, D.M., Juliao, S.F. and Schraw, W.P. (1982) J. Biol. Chem. 257, 11937-11941. 14 Grinnel, F., Hays, D.G. and Minter, D. (1977) Exp. Cell. Res. 110, 175-190. 15 Orten, J.M. and Neuhaus, O.W. (1982) Human Biochemistry, p 441, The C.V. Mosby Company, St. Louis. 16 Okner. R.K., Weisinger, R.A. and Gollan, J.L. (1983) Am. J. Physiol. 245, G13-GI8. 17 Gershenson, L.E., Okigaki, T., Anderson, M., Molson, J. and Davidson, M.B. (1972) Exp. Cell. Res. 71, 49-58. 18 Menezo, Y. and Khatchadourian, C. (1986) Life Sci. 39, 1751-53. 19 Strobel, J.L., Cady, S.G., Borg, T.K., Terracio, L., Baynes, J.W. and Thorpe, S.R. (1986) J. Biol. Chem. 261, 7989-94.