~ ) Pergamon Progress in Growth Factor Research, Vol. 6. Nos. 2~,, pp. 241-251, 1995

Published by Elsevier Science Ltd Printed in Great Britain.

0955-2235/95 $29.00 + .00

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FORMATION OF 150-kDa BINARY COMPLEXES OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN-3 AND THE ACID-LABILE SUBUNIT

IN VITRO AND IN VIVO

C. Young Lee and Mat thew M. Rechler*

Growth and Development Section, Molecular and Cellular Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, MD 20892-1758, U.S.A.

Adult rat serum contains two types of 150-kDa IGFBP complexes: ternary complexes containing bound IGF-I, intact IGFBP-3 and the acid-labile subunit (ALS), and binary complexes that contain ALS and proteolytically-nicked IGFBP-3 but which lack bound IGF. We present evidence that the binary complexes containing proteolytically-nicked IGFBP-3 can be formed in two ways: by direct association of IGFBP-3 with ALS in the absence of lGF, and by proteolysis of lGFBP-3 within 150- kDa ternary complexes, resulting in increased dissociation of IGF-L The relative contributions of the two mechanisms is unknown. Preliminary results indicate that binary complexes also can form in vivo. Proteolysis of 1GFBP-3 in the 150-kDa ternary complex provides a regulatable mechanism by which IGF-I may be mobilized from the circulating reservoir of 150-kDa complexes to the tissues.

Keywords: ALS, IGFBP-3, IGFBP-3 protease.

150-kDa BINARY COMPLEXES OF IGFBP-3 AND ALS ARE ABUNDANT IN ADULT RAT SERUM

Incubation of adult rat serum with radio-iodinated insulin-like growth factor-II (IGF-II) led to the rapid formation of 150-kDa complexes that could be identified by size exclusion chromatography at neutral pH (Fig. 1) [1-3]. Competitive binding assays using the 150-kDa pool, radio-iodinated IGF-II and different concentrations of unlabeled IGF-II and IGF-I, demonstrated that these complexes bound IGF-II with higher affinity than IGF-I (Fig. 1) [2, 3]. This suggested the possibility that adult rat serum contained 150-kDa binary complexes of IGF-binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS), in addition to the ternary complexes containing these two components and IGF-I. (Unlike adult human serum, adult rat serum contains negligible amounts of IGF-II.)

*Correspondence to: M. M. Rechler.

241

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FIGURE 1.

b ' - 201-

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0~ 20 :~

i i

Log Nanograms

i

i i i i

40 do sb Fraction Number

100

80

g 80 O

~ 4O

2O

242 C Y. Lee and M. M. Rechler

[12Sl]IGF-lI binds preferentially to the 150-kDa fractions of adult rat serum. Left: fractiona- tion of adult rat serum. Adult rat serum that had been incubated with 112Sl]IGF-II was fractionated on a Sephadex G-200 column at neutral pH. Radioactivity (open circles) and absorbance (A2s0~) is plotted for each fraction. Right: competitive-inhibition of [12Sl]lGF-II binding to the 150-kDa pool (fractions 30-39, left panel, closed bar). An aliquot of the 150-kDa pool was incubated with 35 pg [l~Sl]IGF-II and the indicated doses of unlabeled IGF-I (O) or IGF-II (O). Bound []2Sl]IGF-II (B) at each dose was determined by a char- coal separation assay, and is expressed relative to the binding in the absence of unlabeled ligand (Bo). (Modified from [3].)

An alternative interpretation of our results was that the radiolabeled IGF-II might be displacing IGF-I from the endogenous 150-kDa ternary complexes. This possibility was excluded by an experiment in which the 150-kDa pooled proteins from adult rat serum were incubated with large amounts of unlabeled IGF-II, frac- tions containing bound IGF re-isolated, and their content of bound IGF-II (exoge- nous) and IGF-I (endogenous) quantitated [3]. The 150-kDa pool to which no IGF-II had been added contained 86 ng/ml IGF-I and no measurable IGF-II. Incubation with IGF-II resulted in a small decrease in IGF-I content (to 63 ng/ml), and an 8-fold greater increase in bound IGF-II to 191 ng/ml. Thus, the minor displacement of endogenous IGF-I from 150-kDa complexes could not account for the much greater binding of exogeneous IGF-II, indicating that the added IGF-II bound to binding sites that were already present and unoccupied in the 150-kDa complexes. It remained to be demonstrated that the IGF-II preferring binding site was present on a different population of 150-kDa IGFBP complexes than those to which endogenous IGF-I was bound (Fig. 2) [3]. This was accomplished by IGF-I affinity chromatography of the 150-kDa IGFBP pool of adult rat serum. Ternary complexes whose IGF-I binding sites were occupied by endogenous IGF-I could not bind to the column and appeared in the 'flow-through', whereas binary complexes whose IGF-binding sites were unoccupied were retained by the column and eluted with acetic acid (eluate). Competitive binding assays demonstrated that IGFBPs in the acid-stripped flow-through had equal affinity for IGF-I and IGF-II, whereas IGFBPs in the eluate bound IGF-II with higher affinity than IGF-I. Thus, adult rat serum contains two distinct populations of 150-kDa IGFBP complexes: unoccupied binary complexes that preferentially bind IGF-II, and ternary complexes occupied by endogenous IGF-I that bind IGF-I and IGF-II with equal affinity.

Affinity crosslinking and ligand blotting were performed on the flow-through and eluate to determine if there was a structural basis for the observed difference in

Binary Complexes of ALS and IGFBP-3 243

100

8O

O

2O

Flow-through

"~ IGF-I

-2 -1 0 1 Log Nanograms

2 -3 -:Z

Eluate

Log Nonograms

FIGURE 2. Competitive inhibition of pZSlllGF-ll binding to IGFBPs in the acid-stripped flow-through (left) and the acid eluate (right) following IGF-I affinity chromatography of the 150~kDa pool of adult rat serum. Samples were incubated with the indicated amounts of unlabeled IGF-I (©) or IGF-II (O). Bound [I~I]IGF-II was determined in a charcoal-separation assay. (Modified from [3].)

IGF-binding specificity (Fig. 3) [3]. When samples were incubated with [125I]IGF-II, cross-linked with disuccinimidyl suberate, and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), predominant 50-kDa complexes and smaller amounts of 37-kDa complexes were observed in both the flow-through (lane 1) and eluate (lane 4). Although the size of these complexes and the fact that they were isolated from the 150-kDa pool strongly suggested that they contained IGFBP-3, this was confirmed directly by immunoprecipitation with specific antiserum. The differences in IGF-binding specificity of the flow-through and eluate IGFBPs again were observed: formation of the complexes with flow- through IGFBPs was inhibited equally by IGF-I and IGF-II, whereas complexes formed by eluate IGFBPs were preferentially inhibited by IGF-II (lanes 1-6). A second and striking difference was observed on ligand blotting: a band correspond- ing to full size 43-kDa IGFBP-3 was seen with the flow-through sample (lane 9), but not with eluate (lane 8), which contained only a 30-kDa doublet. These results indicate that IGFBP-3 in the flow-through is intact, whereas IGFBP-3 in the eluate is proteolytically nicked, consisting of non-covalently linked 30-kDa and smaller proteolytic fragments. The two fragments in the eluate remained closely associated, as they could be covalently cross-linked, but were dissociated under the denaturing conditions of ligand blotting without crosslinking.

Thus, adult rat serum contains two types of 150-kDa complexes: classic ternary complexes of IGF-I, intact IGFBP-3 and ALS, and binary complexes that contain ALS and proteolytically-nicked IGFBP-3, but which lack bound IGF.

150-kDa BINARY COMPLEXES OF ALS AND IGFBP-3 CAN FORM DIRECTLY

The 150-kDa binary complexes of ALS and proteolytically-nicked IGFBP-3 might form in either of two ways: (1) by direct association of ALS and IGFBP-3, or (2) by proteolysis of IGFBP-3 within the ternary complex, with subsequent release of IGF-I.

244 C. Y. Lee and M. M. Rechler

k D a

2 0 0 -

97 .4 -

C r o s s l i n k i n g

wW

L i g a n d Blot

l I B ~ k D a - 2 0 0

- 97 .4

. - 6 9

6 9 -

4 6 -

~ 4 L ~

- 46

- 30

- 21 .5

21 .5 - 1 2 3 4 5 6 7 8 9

- 14 .3

FIGURE 3. Affinity cross-linking (lanes 1-6) and Western ligand blot analysis (lanes 7-9) of the 150-kDa IGFBP pool after IGF-I affinity chromatography. Lanes 1-6; aliquots of the acid-stripped flow-through (lanes 1-3) and the acid-eloate (lanes 4-6) after IGF-I affinity chromatography of the 150-kDa pool, were incubated with 120 pg p~I]IGF-II alone (lanes 1 and 4) or in the presence of 200 ng IGF-I (lanes 2 and 5) or IGF-II (lanes 3 and 6). Complexes were cross-linked using disuccinimidyl suberate, and fractionated by SDS-PAGE under reducing conditions. Lanes 7-9: aliquots of the same samples were analyzed by Western ligand blotting using [nzsI]IGF-II. Lane 7, 150-kDa IGFBP pool before IGF-I affinity chromatography; lane 8, acid-eluate; lane 9, flow-through. (Modified from [3].)

Two experiments were performed that establish convincingly that the first mech- anism, direct association of ALS and IGFBP-3, can occur [4]. Rat ALS was puri- fied in our laboratory by DEAE-Sephadex anion-exchange chromatography followed by affinity chromatography on an IGF-I affinity column to which recom- binant human (h) IGFBP-3 (provided by Celtrix, Santa Clara, CA) was non-cova- lently bound. Incubation of ~25I-labeled rat ALS with intact recombinant human IGFBP-3 in the absence of IGF formed complexes that could be immunoprecipi- tated by antiserum to hlGFBP-3 (also provided by Celtrix) (Fig. 4, lane 4). Any IGF that might have been introduced in the assay reagents including albumin was too low to account for the observed complexes. Complex formation was increased by addition of IGF-I to the incubation (lane 7). Thus, formation of 150-kDa IGFBP complexes can occur in the absence of IGF, but is more efficient when IGF is present.

Binary Complexes of ALS and IGFBP-3 245

oLhlGFBP-3 Non-immune

hlGFBP-3 IGF-I ALS

m

m

n

m

+

n

m

m

- - +

+

- - +

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+

-I- +

_

+ -I-

+ +

+ +

- - +

m

+

.-I-

"-I-

kDa

200 -

9 7 . 4 -

6 8 -

4 3 -

2 9 - -

1 2 3 4 5 6 7 8 9 FIGURE 4. lmmunoprecipitation of [Iz5I]rALS after incubation with hlGFBP-3. [1251]rALS (200,000 c.p.m.) was incubated alone (lanes 2 and 3), or with 10 ng hlGFBP-3 (lanes 4-9) in the absence (lanes 4-6) or presence (lanes 7-9) of 10 ng IGF-I (lanes 7-9) in phosphate-buffered saline (pH 7.4) containing 0.2% BSA for 16 h at 4°C. Unlabeled rALS (1 ~g) was added to lanes 5 and 8. Bound [125I]rALS was immnno- precipitated using rabbit antiserum to hlGFBP-3 (lanes 2, 4, 5, 7, 8) or nonimmune serum (lanes 3, 6, 9). The immunoprecipitates were washed, solnbilized and examined by 10% S D S - P A G E under reducing condi- tions. In lane 1, 1125I]rALS (15,000 c.p.m.) was examined directly without immunoprecipitation. (Reprinted from 1413

In the second experiment, formation of binary complexes of unlabeled rat ALS and recombinant human IGFBP-3 in the absence of IGF was demonstrated by ligand blotting with [t25I]IGF-II following affinity cross-linking (Fig. 5). Cross- linked binary complexes whose IGF-binding sites were unoccupied were identified by their ability to bind [125I]IGF-II after SDS-PAGE and transfer to a nitrocellu- lose membrane (lane 2). The unoccupied sites were present on binary complexes that existed prior to affinity cross-linking, and did not arise by dissociation of IGF- I during SDS-PAGE from ternary complexes that had been covalently cross-linked between ALS and IGFBP-3, but not between IGF-I and IGFBP-3. This was attested by the fact (1) that incubation with exogeneous unlabeled IGF-I prior to cross-linking decreased the binding of radiolabeled IGF-II after SDS-PAGE (lane 3), and (2) that incubation with radiolabeled IGF-I prior to cross-linking increased the total radioactivity associated with the cross-linked complexes following SDS-PAGE and ligand blotting (lane 4, representing cross-linked [125I]IGF-I plus noncovalently bound [125I]IGF-II). This is a compelling result, because the maximum amount of possible contamination by IGF-I was quite low in this exper-

246 C Y. Lee and M. M. Rechler

h l G F B P - 3 + + + + -

r A L S - + + + +

IGF-I [12Sl]IGF-I

4-

-I-

k D a

2 0 0 -

9 7 . 4 -

6 8 -

4 3 -

1 2 3 4 5

FIGURE 5. Ligand blotting of cross-linked complexes of rALS and hlGFBP-3. Glycosylated hlGFBP-3 (10 ng) and rALS (100 rig) were incubated individually, or together in the presence or absence of 10 ng IGF- I or 15,000 c.p.m. 11251]IGF-I as indicated at the top of the figure. After cross-linking using disuccinimidyl soberate, proteins were subjected to 8.5% SDS-PAGE and transferred to a nitrocellulose membrane which was incubated with [IzsI]IGF-II, washed and aotoradiographed. The position of a broad 185-kDa band that was observed when hlGFBP-3 and rALS were incubated together but not separately is indicated by an arrow. The reduced electrophoretic mobility of these cross-linked binary complexes probably results from an inability to uniformly bind SDS due to cross-linking within each subunit. (Reprinted from 14].)

imen t , a n d because any I G F - I p r e sen t in the r e c o n s t i t u t e d c o m p l e x e s w o u l d have

been c ros s - l i nked to I G F B P - 3 a n d d e c r e a s e d [125I]IGF-II b ind ing . A s imi la r resul t

was r e p o r t e d by B a r r e c a et al. us ing h u m a n A L S [5].

Binary Complexes of ALS and IGFBP-3 24 7

Oh 48h

Non-immune Anti-rALS Anti-hBP-3

- - - I -

- I -

- - - I -

- I -

kDa

200 -

97.4 -

6 6 - M

-I-

4 3 -

2 9 -

18.4 -

1 2 3 4 5 6 7

FIGURE 6. Affinity cross-linking and immunoprecipitation of reconstituted ternary complexes containing [~2si]IGF-I after incubation with rat serum using antiserum to rALS or hlGFBP-3. Reconstituted ternary complexes containing [12Sl]IGF-I, hlGFBP-3 and rALS were incubated with rat serum at 37°C for 0 or 48 h. The 150-ki)a complexes were re-isolated by neutral gel filtration, cross-linked with disuecinimidyl suber- ate, and analyzed by 10% SDS--PAGE under reducing conditions either directly (lanes 1 and 5) or after precipitation using non-immune rabbit serum (lane 2), or rabbit antiserum to rALS (lanes 3 and 6) or hlGFBP-3 (lanes 4 and 7).

248 C. Y. Lee and M. M. Rechler

These and other results strongly suggest that ALS and hlGFBP-3 can form binary complexes in the absence of IGF. The IGFBP-3 must be intact. Proteolytically-nicked IGFBP-3 does not bind to rALS in the absence of IGF, and binds poorly even in the presence of the ligand [4].

Preliminary experiments indicate that binary complexes also can form by direct association of IGFBP-3 and ALS in vivo in the rat. In experiments similar to those described by Lewitt et al. [6], and by Zapf et al. [7], we have observed that intra- venously infused non-glycosylated hlGFBP-3 rapidly forms 150-kDa complexes in an abundance comparable to that of pre-existing 150-kDa ternary complexes. To explain the rapid formation of 150-kDa complexes, Lewitt et al. [6], assuming that IGFBP-3 first needed-to bind IGF-I before it could bind to ALS [8, 9], postulated a rapid influx of IGF-I into the circulation after mobilization from an unknown source. If this were the case, the levels of immunoreactive IGF-I in serum should increase dramatically following hlGFBP-3 infusion. We observed no increase in IGF-I content, however, indicating that infused hlGFBP-3 did not need to bind IGF-I in order to form 150-kDa complexes.

150-kDa BINARY COMPLEXES OF ALS AND IGFBP-3 ALSO ARE FORMED BY PROTEOLYSIS OF IGFBP-3 WITHIN 150-kDa TERNARY

COMPLEXES WITH RELEASE OF IGF-I [12]

Proteolysis of IGFBP-3 within 150-kDa complexes was demonstrated using 150- kDa complexes that were reconstituted from recombinant hlGFBP-3, partially purified rALS and [~25I]IGF-I, and isolated by neutral gel filtration. The reconsti- tuted complexes were incubated at 37°C, after which the 150-kDa complexes were re-isolated by neutral gel filtration, affinity-crosslinked, immunoprecipitated with antiserum to rALS (raised in our laboratory), antiserum to hlGFBP-3 or non- immune serum, and the immunoprecipitates examined by SDS-PAGE (Fig. 6). Unincubated samples contained radiolabeled complexes of 95 kDa and larger that were immunoprecipitated by both antisera, and 50-kDa complexes that only were immunoprecipitated by antiserum to hlGFBP-3. Following incubation for 48 h at 37°C, additional complexes of 37, 27 and 23 kDa appeared that were immunopre- cipitated by anti-hlGFBP-3. These results indicate that incubation of 150-kDa complexes containing intact hlGFBP-3 resulted in the partial proteolysis of hlGFBP-3, while it remained as part of the 150-kDa ternary complex with ALS and [1251]IGF-I.

Proteolysis of endogeneous rIGFBP-3 within 150-kDa complexes also was observed (Fig. 7 and [12]). Following incubation of adult rat serum at 37°C followed by ligand blotting, IGFBP bands at 43 and 30 kDa that correspond to intact and truncated IGFBP-3, respectively, decreased in a time-dependent fashion, whereas 29- and 24-kDa IGFBP-4 were not affected. The same decrease in intact and truncated IGFBP-3 was observed when the incubated samples were immuno- precipitated with antiserum against rALS, indicating that all or part of the decrease represented IGFBP-3 associated with ALS in 150-kDa complexes. The decrease in IGFBP-3 could be inhibited by addition of EDTA (Fig. 7) or by serine protease inhibitors [12], suggesting that the decrease resulted from IGFBP-3 protease activity in adult rat serum.

Binary Complexes of ALS and IGFBP-3 249

k D a

- 2 2 0

- 9 7 . 4

- 6 6

- 4 6

- 3 0

- 2 1 . 5

1 2 3

FIGURE 7. Proteolysis of endogenous IGFBP-3. Adult rat serum was incubated for 20 h at -20°C (lane 1) or at 37°C in the absence (lane 2) or presence of 5 mM EDTA (lane 3) followed by ligand blotting using [J25I]IGF-II. The intensity of the IGFBP bands corresponding to intact (43 kDa) and truncated (30 kDa) IGFBP-3 was decreased by incubation in the absence of EDTA; 29-kDa and 24-kDa IGFBP-4 was not affected.

Proteolysis of hlGFBP-3 in reconstituted 150-kDa complexes following incuba- tion with adult rat serum resulted in the redistribution of [~25I]IGF-I radioactivity from the 150-kDa to the 40-kDa peak (Fig. 8). Formation of the 40-kDa radioac- tive peak was largely inhibited by inclusion of EDTA in the incubation, suggesting that it required proteolysis and did not simply represent dissociation from the intact complex. At least some of the 40-kDa complexes represent the association of released [125I]IGF-I with new IGFBPs. In particular, transfer to rlGFBP-3 could be demonstrated by immunorprecipitation of [~25I]IGF-I with an antiserum (kindly provided by Nicholas Ling) that specifically recognizes rat IGFBP-3 but not human

250 C Y. Lee and M. M. Rechler

~ - 1 2 o oOh

20 40 60 80 Fraction Number

FIGURE 8. Dissociation of [125IIIGF-I from reconstituted ternary complexes after incubation with rat serum. Reconstituted ternary complexes containing [12Sl]IGl-! (100,000 c.p.m.), hlGFBP-3 and rALS were incubated with adult rat serum at 37°C for 0 h (C)) or 48 h (O) followed by neutral Sephadex G-100 gel filtration at 4°C. Radioactivity is redistributed from the 150-kDa peak to the 40-kDa peak. The redistrib- ution is inhibited by EDTA, indicating that it is promoted by proteolysis [12]. At least some of the 112Sl]iGF - 1 is transferred from hiGFBP-3 to rlGFBP-3 (unpublished results).

IGFBP-3. It is reasonable to conclude that proteolysis of IGFBP-3 within the 150-kDa complex promotes the release of the iigand by decreasing the affinity of the IGFBP-3 for IGF-I [3, 10]. This mechanism could contribute to the formation of the 150-kDa binary complexes of ALS and proteolytically-nicked IGFBP-3 that have been observed in adult rat serum. We cannot, however, exclude the possibility that some of the [125I]IGF-I was released from the 150-kDa complex in association with hlGFBP-3.

CONCLUSIONS

We have demonstrated that adult rat serum contains two types of 150-kDa IGFBP complexes that contain ALS: ternary complexes that contain bound IGF-I and intact IGFBP-3, and binary complexes that contain proteolytically-nicked IGFBP-3 but which lack bound IGF. The proteolytically-nicked IGFBP-3 can be formed in either of two ways: by direct association of IGFBP-3 with ALS in the absence of IGF (followed by subsequent proteolysis of the IGFBP-3), or by prote- olysis of IGFBP-3 within 150 kDa ternary complexes resulting in increased dissoci- ation of IGF-I. The relative contributions of these two mechanisms is unknown. Preliminary results indicate that binary complexes also can form in vivo. Proteolysis of IGFBP-3 in the 150-kDa ternary complex provides a regulatable mechanism by which IGF-I may be mobilized from the circulating reservoir of 150-kDa complexes to the tissues. Blat et al. [11] have presented evidence that this mechanism may account for the greater bioavailability and mitogenic potency of IGFs in pregnancy serum in which only proteolytically-nicked IGFBP-3 is associated with ALS.

Binary Complexes o f A L S and IGFBP-3 251

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3. Lee CY, Rechler MM. A major portion of the 150-kilodation insulin-like growth factor binding- protein (IGFBP) complex in adult rat serum contains unoccupied, proteolytically nicked IGFBP-3 that binds IGF-II preferentially. Endocrinology 1995; 136: 668-678.

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6. Lewitt MS, Saunders H, Baxter RC. Bioavailability of insulin-like growth factors (IGFs) in rats determined by the molecular distribution of human IGF-binding protein-3. Endocrinology. 1993; 133: 1797-1802.

7. Zapf J, Hauri C, Futo E, Hussain M, Rutishauser J, Maack CA, Froesch ER. Intravenously injected insulin-like growth factor (IGF)I/IGF binding protein-3 complex exerts insulin-like effects in hypophysectomized, but not in normal rats. J Clin Invest. 1995; 95: 179-186.

8. Baxter RC, Martin JL, Beniac VA. High molecular weight insulin-like growth factor binding protein complex. J Biol. Chem. 1989; 264:11,843-11,848.

9. Baxter RC, Martin JL. Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: Determination by reconstitution and affinity-labeling. Proc. Natl Acad Sci USA. 1989; 86: 6898-6902.

10. Lassarre C, Binoux M. Insulin-like growth factor binding protein-3 is functionally altered in preg- nancy plasma. Endocrinology 1994; 134: 1254-1262.

11. Blat C, Villaudy J, Binoux M. In vivo proteolysis of serum insulin-like growth factor (IGF) binding protein-3 results in increased availability of IGF to target cells. J Clin Invest. 1994; 93: 2286-2290.

12. Lee CY, Reehler MM. Proteolysis of insulin-like growth factor (IGF)-binding protein-3 (IGFBP- 3) in 150-kilodalton IGFBP complexes by a cation-dependent protease activity in adult rat serum promotes the release of bound IGF-I. Endocrinology (In press).