Combination therapy of an anticancer drug with the FNIII14 peptide of fibronectin effectively overcomes cell adhesion-mediated drug resistance of acute myelogenous leukemia.

We investigated whether FNIII14, a 22-mer peptide derived from fibronectin (FN) that potently impairs interaction of FN with beta1-integrin, could overcome cell adhesion-mediated drug resistance (CAM-DR) induced by very late antigen (VLA)-4-to-FN interaction in acute myelogenous leukemia (AML). Two AML cell lines, U937 cells and HL-60 cells, and fresh leukemic cells from six AML patients with high alpha4-integrin expression exhibited CAM-DR to cytosine arabinoside (Ara C) through VLA-4-to-FN interaction, while fresh leukemic cells from two AML patients with low alpha4-integrin expression did not display CAM-DR to Ara C. FNIII14 impaired VLA-4-to-FN interaction and restored sensitivity to Ara C in the CAM-DR leukemic cells. In these CAM-DR leukemic cells, upregulation of Bcl-2, which was induced through the focal adhesion kinase/Akt signal pathway upon VLA-4-to-FN interaction, was inhibited by FNIII14 treatment. In a mouse model of minimal residual disease (MRD) in bone marrow, 100% survival was achieved by combining FNIII14 with Ara C, whereas Ara C alone prolonged survival only slightly. The myelosuppression induced by Ara C was not augmented by the combination of FNIII14 in mouse experiments. Thus, the combination of anticancer drugs and FNIII14 holds promise to eradicate MRD in bone marrow after chemotherapy.

The fibronectin-derived antiadhesive peptides suppress the myofibroblastic conversion of rat hepatic stellate cells.

We previously found that fibronectin (FN) had a functional site (YTIYVIAL sequence in the 14th type III module) suppressing the integrin-mediated cell adhesion to extracellular matrix. FN-derived peptides containing this antiadhesive site were also shown to regulate cellular processes such as proliferation, differentiation, and apoptosis. The present study shows that the FN-derived antiadhesive peptides suppress the myofibroblastic conversion of rat hepatic stellate cells (HSC). Freshly isolated HSC underwent myofibroblastic conversion during culture in the presence of FBS, as evaluated by indices representing the phenotypic activation of HSC, including increased proliferation, consumption of vitamin A-enriched lipid droplets, and expression of alpha-smooth muscle actin. However, appearance of these myofibroblastic characters was suppressed by coculturing HSC with the FN-derived antiadhesive peptides. On the other hand, the activated HSC, which had already acquired the myofibroblastic phenotype through repeated subculture, secreted FN and then stimulated matrix assembly of ED-A (+) cellular FN as well as plasma FN, while the FN-derived antiadhesive peptides inhibited them. Furthermore, the FN-derived antiadhesive peptides suppressed the integrin-mediated adhesion of the primary HSC to plasma FN and ED-A (+) cellular FN substrates. These results suggested that the FN-derived antiadhesive peptides down-regulated the myofibroblastic conversion of HSC in an indirect manner by inhibiting the integrin-mediated adhesive interaction of HSC with ED-A (+) cellular FN.

Interaction with heparin and matrix metalloproteinase 2 cleavage expose a cryptic anti-adhesive site of fibronectin.

We recently found that fibronectin (FN) had a functional site [YTIYVIAL sequence in the heparin-binding domain 2 (Hep 2)] that was capable of suppressing the integrin-mediated cell adhesion to extracellular matrix. However, our results also indicated that this anti-adhesive site seemed to be usually buried within the Hep 2 domain structure because of its hydrophobic nature, raising a question as to the physiological significance of the cryptic anti-adhesive activity of FN. The present study demonstrates that the cryptic anti-adhesive activity can be exposed through the physiological processes. A 30-kDa chymotryptic FN fragment derived from Hep 2 domain (Hep 2 fragment), which had no effect on adhesion of MSV-transformed nonproducer 3T3 cell line (KN(7)8) to FN, expressed the anti-adhesive activity after treatment with 6 M urea. Light scattering and circular dichroism measurements showed that the urea treatment induced the conformational change of the Hep 2 fragment from a more compact form to an unfolded one. Incubation of the Hep 2 fragment with heparin also induced similar conformational changes and expression of anti-adhesive activity. Additionally, both the urea and heparin treatments made the Hep 2 fragment and intact FN much more accessible to the polyclonal antibody (alphaIII14A), with a recognition site near the anti-adhesive site of FN. Specific cleavage of either the Hep 2 fragment or intact FN by matrix metalloproteinase 2 (MMP-2) released a 10-kDa fragment with the anti-adhesive activity, which was shown to have the exposed anti-adhesive site on the amino-terminal region. Thus, the cryptic anti-adhesive activity of FN can be expressed upon conformational change and proteolytic cleavage of Hep 2 domain.

The fibronectin-derived anti-adhesive peptide III14-2 suppresses adhesion and apoptosis of leukemic cell lines through down-regulation of protein-tyrosine phosphorylation.

We previously found that fibronectin (FN) has a cryptic functional site (YTIYVIAL, #1848-1855) opposing to cell adhesion to extracellular matrix (ECM). The present study demonstrates that the FN peptide containing this anti-adhesive site, termed peptide III14-2, affects programmed cell death (PCD) (apoptosis) as well as cell adhesion by down-regulating protein-tyrosine phosphorylation. Peptide III14-2 suppressed the integrin alpha5beta1-mediated adhesion of leukemic cell lines (K562 and HL60), and protein tyrosine phosphatase inhibitor, 1 microM phenylarsine oxide (PAO) blocked the anti-adhesive effect of peptide III14-2. These leukemic cells underwent PCD when exposed to PAO at the higher concentration (5 microM), as judged by nuclear and DNA fragmentations, and which was reversed by tyrosine kinase inhibitor, genistein. Peptide III14-2 suppressed the PAO-induced PCD, whereas a control peptide in which the anti-adhesive sequence YTIYVIAL is scrambled, was inactive. Western blotting showed that PAO stimulated the tyrosine phosphorylation of cellular proteins including focal adhesion kinase and that peptide III14-2 inhibited them, suggesting that protein-tyrosine phosphorylation represents a common early signal for the adhesion and PCD. The anti-adhesive site of FN molecule may play a crucial role also in a variety of cellular processes other than adhesion and PCD by down-regulating protein-tyrosine phosphorylation.

Modulation of apoptotic cell death by extracellular matrix proteins and a fibronectin-derived antiadhesive peptide.

Cell adhesion to the extracellular matrix (ECM) has been implicated in apoptosis in anchorage-dependent cell types. We recently found that a peptide derived from fibronectin (termed III14-2) inhibits the integrin-mediated cell adhesion to ECM. Using this antiadhesive peptide and a variety of ECM proteins, we show here a critical role of the integrin-ECM protein interaction in apoptotic regulation of human umbilical vein endothelial cells (HUVEC). HUVEC in suspension underwent apoptosis under the serum-free conditions, as judged by nuclear and DNA fragmentations. This apoptosis was suppressed to varying degrees when alpha 5 beta 1, alpha v beta 3, and alpha 2 beta 1 integrins were occupied with either soluble or immobilized ECM proteins such as fibronectin, vitronectin, and type I collagen, respectively. Peptide III14-2, which had no effect by itself on the HUVEC apoptosis, disrupted the ligation of alpha 5 beta 1 and alpha v beta 3 but no alpha 2 beta 1 and ultimately led the cells to apoptosis, indicating that this antiadhesive peptide indirectly induces apoptosis by blocking cell survival signal delivered from alpha 5 beta 1 and alpha v beta 3 integrins. Genistein, a protein tyrosine kinase inhibitor, slightly reduced the rescuing effect of fibronectin, whereas sodium orthovanadate and bombesin, which increase in the level of protein tyrosine phosphorylation, made HUVEC less susceptible to apoptosis and blocked the effect of peptide III14-2. HUVEC adhesion to fibronectin substrate raised the tyrosine phosphorylation level of focal adhesion kinase and the expression of cytoprotective Bcl-2 protein, both of which were reversed by the antiadhesive effect of peptide III14-2. Thus, the opposing effects of ECM proteins, including fibronectin and vitronectin, and peptide III14-2 on HUVEC apoptosis appear to be due to the opposing effects of these factors on the signaling pathway which includes tyrosine phosphorylation of FAK and Bcl-2 expression.

Characterization of fibronectin-related substances in normal and passive Heymann nephritis rats.

The excretion mechanism of fibronectin (FN)-related substances into the urine of normal and passive Heymann nephritis (PHN) rats was studied using enzyme immunoassay and immunoblot analysis. In normal rats, a small amount (0.20+/-0.067 microg/d) of FN-related substances, composed of 55- and 65-kDa FN fragments derived from the central cell-binding (Cell) domain of FN, were constitutively excreted into the urine. When PHN was induced in rats by the injection of an anti-Fx1A antibody, an increased excretion (4.96+/-3.51 microg/d) of intact FN and large (Mr > 100-kDa) FN fragments containing the Cell and the other functional domains were seen. The PHN induction also caused the appearance of a considerable amount of Cell domain-containing FN fragments in plasma. Both the renal cortex homogenates of normal and PHN rats were capable of degrading plasma FN to generate the Cell domain-containing large FN fragments. Degradation of FN by the renal cortex homogenate was shown to be due to metal and/or thiol proteinase(s). These results suggest that the PHN-induced urinary excretion of FN fragments may be due to the degradation of plasma FN by renal proteinases that may be leaked upon PHN induction.

Mesangial cell apoptosis induced by a fibronectin fragment.

We previously showed that in passive Heymann nephritis (PHN) rats, a large quantity of fibronectin (FN) fragments containing the central cell-binding (CCB) domain and adjacent domains are generated in the kidney and excreted into urine (Nishizawa et al., Biol Pharm Bull 1998; 21: 429-433). To ascertain whether the FN fragments could affect the progression of PHN, we investigated the effect of a 150 K FN fragment containing the CCB and carboxyl-terminal heparin-binding (Hep 2) domains on cultured rat mesangial cells. When rat mesangial cells cultured on FN-coated plates were exposed to the 150 K FN fragment, some mesangial cells detached from the FN substrate and then underwent apoptosis as judged by nuclear and DNA fragmentations. The 150 K FN fragment competitively inhibited the mesangial cell adhesion to the FN substrate in a dose-dependent manner. Furthermore, gelatinzymography of the conditioned medium of mesangial cells showed that the 150 K FN fragment induced and/or potentiated the extracellular matrix (ECM)-degrading proteinases including matrix metalloproteinases (MMPs) of mesangial cells. These results indicate that the 150 K FN fragment may elicit mesangial cell apoptosis by disrupting the mesangial cell adhesion through two distinct ways: the inhibition of mesangial cell adhesion and the ECM-degradation by the 150 K FN fragment-induced MMPs. Thus, FN fragments containing the CCB and adjacent domains generated in the kidneys of PHN rats may be involved in the evolution of the renal injury.

Identification of the anti-adhesive site buried within the heparin-binding domain of fibronectin.

We recently found that heparin-binding domain 2 (Hep 2) of fibronectin (FN) exhibits cryptic anti-adhesive activity. In order to locate the anti-adhesive site, a number of synthetic peptides which represents the primary structure of the Hep 2 domain were characterized as to their ability to decrease the adhesion of A375SM melanoma cells to FN substrate. Only one peptide (T-E-A-T-I-T-G-L-E-P-G-T-E-Y-T-I-Y-V-I-A-L, residues 1835-1855) (peptide III14-2), which is situated between the previously identified adhesive sites, FN-C/H-I and II, decreased the cell adhesion to FN. Assaying of the anti-adhesive activities of sub-peptides showed that the hydrophobic moiety of peptide III14-2 (underlined sequence) seems to be indispensable for the anti-adhesive activity. These results suggest that anti-adhesive activity is closely associated with the sequence, Y-T-I-V-I-A-L, that is usually buried within the Hep 2 domain structure because of its hydrophobic nature.

Transcellular biosynthesis of cysteinyl leukotrienes by Kupffer cell-hepatocyte cooperation in rat liver.

We previously proposed that an enzymatic cooperation between Kupffer cells and hepatocytes may play an important role in cysteinyl leukotriene (LT) production in rat liver. An in vitro transcellular synthesis cysteinyl LTs by a Kupffer cell-hepatocyte coculture system was characterized here. Kupffer cells alone, with A23187 stimulation, did not generate cysteinyl LTs until supplemented either with isolated hepatocytes or with LTC4 synthase and glutathione, indicating that Kupffer cells can synthesize LTA4 but not convert it into LTC4. In contrast, hepatocytes converted the LTA4 into cysteinyl LTs and further degraded the cysteinyl LTs. Cysteinyl LT production by the Kupffer cell-hapatocyte coculture system was optimized by addition of 1-3% serum albumin to the culture and by bringing the cell-cell distance closer to less than 3 mu. Tumour necrosis factor also stimulated cysteinyl LT production by the coculture system. From these results, it is expected that the Kupffer cell-hepatocyte transcellular system for cysteinyl LT production actually functions in vivo.

Randomized controlled trial of acellular diphtheria, pertussis and tetanus vaccines in southern Ghana.

A randomized controlled trial of acellular diphtheria/pertussis/tetanus (ADPT) freeze-dried and liquid vaccines in infants was conducted in a peri-urban community (Ashaiman) in southern Ghana. Immunogenicity of the acellular vaccines, persistence of antibodies and adverse reactions were compared with those achieved with a whole-cell diphtheria-pertussis-tetanus (DPT) vaccine. The incidence of pertussis in the vaccine groups and prevalence of pertussis in children under 5 years of age in the study area were also determined. The acellular vaccines produced significantly fewer local and systemic reactions. Local reactions such as swelling and redness were observed in 2% (8/399) to 2.3% (9/385) of the acellular vaccine recipients as against 31% (122/394) in the whole-cell vaccine group. Fever ( > or = 37.5 degrees C) occurred in 7.27% (29/399) to 9.8% (38/385) in the acellular vaccine groups compared with 36.6% (145/394) in the whole-cell vaccine group. Geometric mean titres (GMTs), measured by ELISA, to pertussis toxin (PT) and filamentous haemagglutinin (FHA) were significantly higher in the acellular vaccine groups than in the whole-cell DPT (WCDPT) group. There were no significant differences in the GMTs of tetanus and diphtheria antitoxins between the two groups after each vaccination. Twelve months after primary vaccination, GMTs to PT in the freeze-dried, liquid ADPT groups and the WCDPT group have fallen from 56.23, 62.63 and 44.97 ELISA U/ml to 6.08, 6.18 and 11.30 ELISA U/ml, respectively. GMTs to FHA in all the vaccine groups also dropped during the same period from 49.94, 41.73 and 20.74 ELISA U/ml to 7.26, 7.72 and 5.91 ELISA U/ml, respectively. In this comparative controlled trial, the ADPT vaccines were more immunogenic, with less local and systemic reactions, than the WCDPT vaccine but there was a considerable drop in antibody titres in all the vaccine groups 12 months after primary vaccination. However, the levels of titres of anti-PT and anti-FHA antibodies in all the three vaccines that confer protection are not known. Further studies are necessary to provide this information in order to assess the need for subsequent booster doses after primary immunization with both ADPT and WCDPT vaccines.

Fibronectin harbors anticell adhesive activity.

Effect of fibronectin (FN) fragment derived from the carboxyl-terminal heparin-binding (Hep 2) domain on cell adhesion was studied. A 30-kDa Hep 2 fragment showed no significant effect on adhesion of Mardin Darbyn caine kidney (MDCK) cells to FN substrate, whereas after exposure to urea, the Hep 2 fragment suppressed the MDCK cell adhesion to FN in an incompetitive fashion. This anti-cell adhesive Hep 2 fragment preferentially inhibited the RGD (Arg-Gly-Asp)-dependent cell adhesion to FN. No significant binding of the Hep 2 fragment with FN was detected. Additionally, data of flow cytometry showed the presence of specific binding site for the Hep 2 fragment on MDCK cell surface. These results indicate that the Hep 2 domain of FN has a cryptic molecular region having the anti-cell adhesive activity that is probably transduced by a putative cell surface receptor.

Release of biological activities from quiescent fibronectin by a conformational change and limited proteolysis by matrix metalloproteinases.

We reported that specific biological activities are confined to three domains of the fibronectin (Fn) molecule [Fukai et al. (1991) J. Biol. Chem. 266, 8807; Fukai et al. (1993) Biochemistry 32, 5746]: the potent ability to stimulate the adipocyte differentiation of ST-13 cells is in the amino-terminal fibrin-binding (Fib 1) domain (referred to as Fib 1 domain activity); the RGD-dependent activities that stimulate NIH-L13 cell migration and inhibit adipocyte differentiation are in the central cell-binding (Cell) domain (Cell domain activity); and the activity that stimulates cell migration in a RGD-independent manner is in the carboxyl-terminal fibrin-binding (Fib 2) domain (Fib 2 domain activity). Human plasma Fn which was purified without exposure to a denaturant, such as urea, exhibited no Fib 1, Fib 2, or Cell domain activity. By exposure to urea or surface adsorption, Fn showed Cell domain activity but not those of the Fib 1 and Fib 2 domains. Whether the cryptic domain activities are disclosed or not depended on whether or not the responsible domains were irreversibly exposed from confined environments of Fn structure as confirmed by light-scattering measurement and enzyme immunoassay using domain-specific monoclonal antibodies. We then investigated the action of matrix metalloproteinases (MMPs) in liberating the Fib 1, Cell, and Fib 2 domain activities. Matrilysin released only the Cell domain activity. In contrast, stromelysin, collagenase, and especially gelatinase A additionally liberated the Fib 1 and Fib 2 domain activities. The Fib 1, Fib 2, and Cell domains acquired much higher activities when they were freed from linkage with adjacent domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat hepatocytes generate peptide leukotrienes from leukotriene A4.

The biosynthetic mechanism of peptide leukotrienes was studied in rat liver. A catalytic activity to synthesize leukotriene (LT) C4 from LTA4 (LTC4 synthesis activity) was shown to be associated mainly with the microsomal fraction of hepatocytes, but also to a small extent with nonparenchymal cells including Kupffer cells, suggesting that the hepatocytes have an ability to generate peptide LTs. Stimulation of the isolated hepatocytes with calcium ionophore (A23187) did not cause any significant production of peptide LTs, whereas addition of LTA4 induced a remarkable generation of peptide LTs in a dose-dependent manner. Addition of LTA4 also augmented the peptide LT production by Kupffer cells, but its amount was much smaller than that by the hepatocytes under the same culture conditions. Coincubation of the hepatocytes and Kupffer cells, following A23187 stimulation, elicited a markedly enhanced production of peptide LTs even without any addition of LTA4, whereas the LT production in the coincubation system was suppressed almost completely by treating the Kupffer cells with a specific inhibitor of 5-lipoxygenase, AA861 (10 microM). An enzymatic cooperation between the hepatocytes and Kupffer cells may play an important role in generating peptide LTs in the liver.

An amino-terminal fibronectin fragment stimulates the differentiation of ST-13 preadipocytes.

Differentiation of ST-13 preadipocytes into adipocytes was inhibited almost completely by addition of rat plasma fibronectin (FN) (approximately 100 micrograms/mL), but was reversed by GRGDSP cell recognition peptide (1.5 mM) and anti-alpha 5 beta 1. On the contrary, the thermolysin digest of FN stimulated adipocyte differentiation in a dose-dependent manner, in which remarkable increases in the values of the differentiation indexes, the number of adipocytes (8-fold above the control), glycerophosphate dehydrogenase (GPD) activity (12-fold), and triacylglycerol content (5-fold), were observed by inclusion of the thermolysin digest (100 micrograms/mL). The increase in GPD activity by the thermolysin digest was inhibited remarkably (about 70% inhibition) by an antibody directed to the amino-terminal fibrin-binding (Fib 1) domain of FN and slightly (about 15%) by an antibody directed to the central cell-binding (Cell) domain, but not by anti-gelatin-binding domain and anti-carboxy-terminal fibrin-binding domain. Treatment of ST-13 cells by a purified 24K fragment (100 micrograms/mL) derived from the Fib 1 domain caused an over 20-fold augmentation of the GPD activity, accounting for a major part of the differentiation stimulatory activity of the thermolysin digest. The differentiation stimulatory effect of the 24K Fib 1 fragment was not affected by either GRGDSP peptide or anti-alpha 5 beta 1. Thus, FN can regulate adipose development of ST-13 cells by its two antipodal, inhibitory and stimulatory, activities, the latter of which is expressed only upon fragmentation. Proteolytic cleavage of FN may play an important role in controlling the action of FN on adipocyte differentiation.

Involvement of calcium-dependent cysteine protease in fibronectin-induced chemotactic migration of NIH-L13 fibroblasts.

Fibronectin (FN)-induced chemotactic migration of NIH-L13 fibroblasts was suppressed by the inhibitors of cysteine proteases, leupeptin (IC50 = 3 microM), and the epoxysuccinyl derivatives [E-64c (10 microM), SUT-8801 (45 microM), and SUT-8818 (28 microM)], but hardly by phenylmethanesulfonyl fluoride (> 1 mM) and 1-tosyl-amido-2-phenylmethyl chloromethyl ketone (> 1 mM). E-64d, a membrane-permeant derivative of E-64c, was much more effective (IC50 = 4 microM) than E-64c in preventing the cell migration. Furthermore, the FN-induced cell migration was blocked partially with EGTA and was reversed by addition of Ca2+ ion. These results suggest that intracellular Ca(2+)-dependent cysteine proteases may be involved in the FN-induced chemotactic migration of NIH-L13 fibroblasts.

Fibronectin stimulates protein synthesis in cultured fibroblastic cells.

Protein synthesis in mouse embryo fibroblastic (NIH-L1) cells was stimulated by addition of fibronectin (FN) to the culture medium as a soluble factor. This stimulatory activity of FN appeared only when the cells were cultured over their saturated density. A hexapeptide (GRGDSP) containing the RGDS cell attachment site of FN could not prevent the stimulation of protein synthesis. Moreover, activity of the stimulatory FN was not affected by inclusion of cytochalasin B, which is known to release FN from cell surface by disorganizing actin cytoarchitecture. These results suggest that the signaling of change in protein synthesis rate by FN may be distinct from signaling involving cell attachment and spreading.

Stimulation of the protein synthesis by fibronectin is specific for untransformed fibroblastic cells.

In the preceding paper, we showed that fibronectin (FN) in solution has the ability to stimulate protein synthesis in NIH-L1 fibroblastic cells. We show here that FN stimulated protein synthesis also in other normal fibroblast cell lines, WI-38 and BALB/3T3, but did so only slightly in their transformed cell lines, L1-4NQOH10, WI-38 VA-13 and KN(7)8; and in rat hepatocytes in primary culture and rat liver epidermoid cells (GR-1) it hardly affected the synthesis. Augmentation of the protein synthesis rate was not attributable to stimulation of collagen synthesis. This stimulatory activity of FN was enhanced remarkably by proteolytic digestion of FN with trypsin.

Rat plasma fibronectin contains two distinct chemotactic domains for fibroblastic cells.

Mechanism of fibronectin (FN)-induced chemotaxis of fibroblastic cells has not been fully understood. The present study was performed to establish a molecular nature of the chemotactic region of rat plasma FN. The chemotactic dose-response pattern of intact FN for mouse embryo fibroblastic cells, NIH-L13 cells, which was represented as a "bell-shape" curve with a maximum activity at around 50 nM, changed to a "biphasic" mode through a proteolysis with thermolysin. Two distinct chemotactic components were isolated from the thermolytic fragments. One component, a fragment with a molecular mass of 110-150 kDa, was estimated to contain the central cell-binding domain and the carboxyl-terminal heparin-binding domain of the intact FN molecule. Cell migration stimulated by the 110-150-kDa fragment increased successively in a dose-dependent manner, and the capability to promote the migration was much higher than that of the intact FN (over 2-fold). The second chemotactic component, a fragment with a molecular mass of 21 kDa, was shown to reside in the carboxyl-terminal fibrin-binding domain. The 21-kDa fragment produced a bell-shape dose-response pattern, being consistent with the intact FN, whereas a maximum response occurred at a 100-fold lower concentration (0.5 nM) than that of the intact FN molecule. At higher concentrations, this fragment revealed an inhibitory activity for the cell migration in response to the 110-150-kDa fragment. No significant molecular interaction between these two active components was observed by polyacrylamide gel electrophoresis under nondenaturing conditions, suggesting that the 21-kDa fragment may act directly on the cell to inhibit the cell migration. These results suggest that rat plasma FN contains at least two chemotactically active components that regulate cooperatively chemotactic migration of fibroblastic cells.

Chromatographic method for the preparation of apo-cellular retinol-binding protein and apo-cellular retinoic acid-binding protein from their holo-types.

A chromatographic method is described for the preparation of apo-cellular retinol-binding protein (CRBP) and apo-cellular retinoic acid-binding protein (CRABP) from their corresponding holoproteins. Elimination of retinoids from either purified CRBP or CRABP holoprotein complex could be performed quantitatively by DEAE-cellulose chromatography without any alteration in the inherent properties of the native proteins. In contrast, the usual methods, involving UV irradiation or acetone precipitation, resulted in some modification of these binding proteins. This chromatographic method was also applicable to the preparation of apo-fatty acid-binding protein (FABP) from FABP-palmitic acid holoprotein complex.