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Identification of Liver Growth Factor As an Albumin-Bilirubin Complex

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Identification of Liver Growth Factor As an Albumin-Bilirubin Complex Biochem. J. (1987) 243, 443-448 (Printed in Great Britain) 443 Identification of a liver growth factor as an albumin-bilirubin complex Juan J. DIAZ-GIL,*§ Jose G. GAVILANES,t Gonzalo SANCHEZ,* Rafael GARCIA-CANERO,* Juan M. GARCIA-SEGURA,t Luis SANTAMARIA, Carolina TRILLA* and Pedro ESCARTIN* *Servicios de Bioqu;nica Experimental y Gastroenterologia, Clinica Puerta de Hierro, 28035 Madrid, Spain, tDepartamento de Bioquimica, Facultad de Ciencias, Universidad Complutense, 28040 Madrid, Spain, and IDepartamento de Morfologia, Universidad Autonoma, 28029 Madrid, Spain We have reported the purification and characterization of a protein that behaves as a liver growth factor, showing activity either in vivo or in vitro [Diaz-Gil et al. (1986) Biochem. J. 235, 49-55]. In the present paper, we identify this liver growth factor (LGF) as an albumin-bilirubin complex. This conclusion is supported by the results of chemical and spectroscopic characterization of this protein as well as by experiments in vivo. Incubation of albumin isolated from normal rats with bilirubin at several bilirubin/albumin molar ratios (r) resulted (when r = 1 or 2) in a complex with liver DNA synthesis promoter activity identical with that of LGF. The exact amount of bilirubin bound to albumin was assessed by fluorescence and c.d. spectra. This albumin-bilirubin complex showed the same dose-dependence profile as LGF either at low or high dose of protein injected per mouse. Both LGF and albumin-bilirubin complex produced similar increases in the mitotic index of mouse hepatocytes in vivo. A new mechanism for the onset of the hepatic regenerative process is proposed. INTRODUCTION described (Diaz-Gil et al., 1986a). Commercial rat serum albumin (fraction V from Sigma) was purified by a We recently reported the purification of a 64000-Mr three-step procedure very similar to that used for LGF protein from plasma of partially hepatectomized rats purification: chromatography on Sephadex G-75, which exhibits hepatic promoter activity both in vivo and DEAE-cellulose and hydroxyapatite. We refer to this as in vitro (Diaz-Gil et al., 1986a). The injection of this purified serum albumin. Following the same strategy, we protein into mice at nanogram doses (up to purified albumin from plasma of either normal or 150 ng/mouse) increases [3H]thymidine uptake by liver hepatectomized rats. We call these fractions F 121(N) DNA, producing an increase in the mitotic index of and F 121(PH), respectively. hepatocytes. In primary liver cell cultures, it produces an The determination of the activity in terms of DNA increase in the uptake of [3H]thymidine into DNA in the synthesis promoter was carried out in mice, as previously range of 1-10 ng/ml, as well as an increase, immediately indicated (Diaz-Gil et al., 1986a). For the extraction of upon addition, in the uptake of 22Na+. In view of the DNA, the method of MacManus et al. (1972) was used; activity exhibited by this protein and to avoid confusing total DNA was determined by the method of Burton its context with other areas of research, we will refer to (1968). Specific incorporation of [3H]thymidine into this protein hereafter as 'liver growth factor' (LGF). DNA was expressed as d.p.m./,g of DNA. Protein The present paper deals with the characterization of determination was performed by the method of Lowry this LGF. et al. (1951). For experiments in vitro, hepatocytes from 150 g MATERLILS AND METHODS bodywt. Wistar rats were isolated by a collagenase perfusion technique (Bonney et al., 1974). More details Materials regarding the procedure followed to evaluate LGF Chemicals were purchased from Sigma, Bio-Rad, activity are given in a previous publication (Diaz-Gil Cultek, Merck and Pharmacia. [3H]Thymidine (specific et al., 1986a). radioactivity 20 Ci/mmol) was from New England Nuclear. Spectroscopic measurements Wistar rats (90-110 g body wt.) were 70% hepatecto- Absorbance was determined on a Cary 118 spectro- mized by the method of Higgins & Anderson (1931). photometer at a 0.2 nm/s scanning speed on an auto slit Plasma was obtained from heparinized blood of normal program. C.d. spectra were measured in a Jobin Yvon or hepatectomized rats by cardiac puncture. Plasma Mark III dichrograph, fitted with a 250 W xenon lamp, samples were freeze-dried and kept at 4 °C until use. at a 0.2 nm/s scanning speed; 0.05 cm optical path cells were used in the far-u.v. region whereas 1 cm cells were LGF purification and analysis of activity employed in the near-u.v. C.d. results are the average of The purification of LGF was carried out as previously at least five determinations. The mean residue weight for Abbreviations used: F 121(N), albumin from plasma of normal rats; F 121(PH), albumin from plasma of partially hepatectomized rats; LGF, liver growth factor; r, bilirubin/albumin molar ratio. § To whom correspondence and reprint requests should be addressed. Vol. 243 444 J. J. Diaz-Gil and others calculations was 111.5, based on the amino acid Table 1. Amino acid composition of the liver growth factor; composition of the protein (see below). Fluorescence values for rat serum albumin are also included for spectra were obtained on a Perkin-Elmer MPF 44E comparison spectrofluorimeter. Spectra were corrected for detector response. Values are expressed as the nearest integer value based on The protein solutions were filtered through a Millipore an Mr of 65000 for both proteins. For each hydrolysis filter (0.5 gsm pore diameter) prior to the spectroscopic time, three separate determinations were performed. analysis, which was performed at 20 'C. The protein Serine and threonine contents were corrected to zero concentration for the spectroscopic calculations was hydrolysis time. Valine and isoleucine contents were determined from the 96-h hydrolysates. Half-cystine was determined by amino acid analysis. determined as cysteic acid after oxidation of the protein Other analytical procedures samples with performic acid at -10 °C (Hirs, 1956). SDS/polyacrylamide-gel electrophoresis was perform- ed according to Laemmli (1980). For mitotic index Composition (residues/Mr 65000) determination, the procedure of Morley & Kingdon Amino Purified (1973) was employed. The Ouchterlony immunodiffusion acid LGF albumin and immunoelectrophoresis experiments were carried out on agarose (1.5% solution with 35 mM-barbital Cys 32 31 buffer), using 65 mM-barbital, pH 8.6, as running buffer. Asx 47 46 Performic acid oxidized proteins (Hirs, 1956) were Thr 33 35 digested with trypsin at 100:1 protein: enzyme weight Ser 27 28 ratio for 4 h at 37 'C in 0.2 M-NH4HCO3, pH 8.0. The Glx 75 76 resulting peptides were separated by using an Ultrasphere Pro 29 28 ODS column (4.5 mm x 250 mm; 5 #m particle size). Gly 27 23 The protein samples were hydrolysed at 108 'C (for 24, Ale 61 63 48 and 96 h) with constant-boiling HCI containing 0.1 % Val 35 36 (w/v) phenol, in evacuated and sealed tubes. Hydro- Met 6 6 lysates were analysed on a Durrum model D-500 Ile 16 15 amino Leu 54 54 acid analyser. Tyr 21 21 Albumin-bilirubin complex formation Phe 27 27 His 16 16 Bilirubin was purchased from Signa. Its purity was Lys 49 49 checked by the procedure of McDonagh & Assisi (1971). Arg 29 29 9 7 5 3 ;1 x o 9 7 5 3 0 5 15 25 35 45 55 65 75 85 95 105 115 125 135 Time (min) Fig. 1. Reversed phase h.p.l.c. of tryptic digests of performic-acid-oxidized LGF (a) and purified serum albumin (b) Samples (100 jug in 20 ll) were applied in 0.2 M-NH4HCO3, pH 8.0. The column was eluted at 60 ml/h: 10 mM-ammonium acetate, pH 6.5, for 8 min; a linear gradient of 0-2% acetonitrile in the acetate buffer for 10 min; a linear gradient of 2-9% acetonitrile in the same buffer for 28 min; 9% acetonitrile in acetate for 10 min; a linear gradient of 9-15% acetonitrile in the acetate buffer for 24 min; and a linear gradient of 15-35% acetonitrile in acetate buffer for 60 min. Detection of the peptides was performed by measurement of A214. See the Materials and methods section for more details. 1987 Identification of a liver growth factor 445 Binding of bilirubin to albumin was carried out by the exhibits liver growth factor activity both in vivo and method of Wooley & Hunter (1970). In experiments in vitro. The chemical analysis of F 121(PH) revealed where bilirubin was injected alone, it was previously the identity between this protein and LGF. Thus, we will incubated in the same conditions described above for refer to both of them simply as LGF. All of these bilirubin-albumin samples. observations corroborate the supposition that LGF is a special kind of albumin present in plasma of partially hepatectomized rats. RESULTS Serum albumin-bilirubin complex prepared in vitro is a Protein-chemical similarities between LGF and serum liver growth factor albumin Another feasible explanation is that the LGF isolated The amino acid compositions ofalbumin and LGF are could simply be albumin with a very-low-Mr ligand, X. very similar (Table 1). The similarity between the two As a further development of this hypothesis, the proteins is also observed by comparing their tryptic maps hypothetical X ligand could be the true liver growth (Fig. 1). The u.v.-absorbance spectra of purified serum factor, appearing in situations of partial hepatectomy, albumin and LGF are coincident. The fluorescence and absent from normal rats. We considered the emission spectra for excitation at 295 nm are almost possibility that bilirubin be responsible for the liver coincident.
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