S.T. Ding and M.S. Lilburn
Department of Animal Sciences
A purified prep of liver cytosolic fatty acid-binding protein (FABP) was used as a standard for quantitating FABP using two procedures: 1) an immunoblot assay using chick liver FABP antisera and 2) a ligand binding assay using gel filtration and binding of radiolabelled oleic acid. Specific activity of liver FABP (L-FABP; ng/mg cytosolic protein) increased with length of incubation. An initial peak was observed on day 22, followed by a decline (day 25) and subsequent increase through hatching (day 28) and early posthatch growth. FABP concentrations in the intestine and yolk sac membrane also were measured with the immunoblot and ligand binding assays. The chick liver antisera did not cross-react with yolk sac membrane FABP (YS-FABP), and detectable concentrations of FABP in the intestine (I-FABP) were not observed until hatch (day 28). The ligand binding assay confirmed that I-FABP concentration was very low prior to hatch and increased sharply thereafter, probably in response to dietary fatty acids after hatching. YS-FABP activity as determined by ligand binding assay (dpm/mg yolk sac membrane cytosolic protein) increased from day 13 of incubation to a plateau between days 16 and 19, followed by a decline through hatch. Concentrations in the yolk sac membrane correlated well with the period of maximum lipid transfer from the yolk to the developing embryo via the yolk sac membrane.
Small proteins (12 to 15 kd) with high binding affinities for long-chain fatty acids (LCFA) have been identified in many tissues. Collectively, these proteins are called fatty acid-binding protein (FABP) and are involved in intracellular fatty acid transfer (Tipping and Ketterer, 1981). Intestinal FABP is thought to be involved in dietary fatty acid absorption and utilization (Ockner et al., 1972; Ockner and Manning, 1982). In rats and probably other species as well, there are distinct, tissue-specific FABP's (i.e., intestinal FABP, liver FABP, cardiac FABP; Ockner and Manning, 1982; Bass et al., 1985; Shields et al., 1986).
In rats, liver FABP (L-FABP) increases 20-fold from 5 days prior to birth through 45 days of age (Sheridan et al., 1987). In pigs, L-FABP is constant from 73 days of pregnancy through parturition (114 days) and then declines through 7 days of age (Reinhart, 1990; Chi, 1993).
While the tissue specific forms of FABP are distinct proteins, there can be multiple expression of more than one FABP in a specific tissue. For example, in rat embryos both L-FABP and I-FABP are expressed in the intestine as early as 18 days of gestation, but L-FABP expression occurs earlier than I-FABP.
Scapin et al. (1988) purified a basic form of chicken L-FABP (16.5 kD; pI 9.0), while Sewel et al. (1989) characterized a different chick cytosolic FABP (14 kD; PI 7.0). Katangole and March (1979) reported I-FABP activity in chick intestine during the first week of life but found it to be low compared with later ages. There have been no reports to date on FABP activity in liver or intestine from avian embryos. The largest proportion of yolk lipids mobilized from the yolk sac to the embryo occurs during the last week of incubation (Ding et al., 1995; Ding and Lilburn, 1996). While there has been much published relative to the contributions of the yolk sac membrane to avian embryonic lipid metabolism, the existence of FABP in this tissue has not been studied.
Traditional methods for quantifying FABP activity have incorporated either post-column ligand binding assays using radiolabelled fatty acids or some type of immunoblot assay using protein specific antisera. This report will cover the measurement of FABP in embryonic tissues using both a ligand binding assay and an immunoblot assay using a polyclonal antisera generated against chick liver FABP (L-FABP).
Livers, intestine, and yolk sac membrane were collected from turkey embryos and newly hatched poults. The samples were collected beginning at 13 days of incubation (hatch = 28 days). The tissues were immediately washed in ice-cold buffer and snap-frozen in liquid nitrogen. Each tissue subsequently was thawed, homogenized, and the cytosolic fraction collected by centrifugation.
The immunoblot assay was a modification of the procedure of Burnette (1981). After electrophoretic separation and transfer to nitrocellulose, each membrane initially was incubated with a 1:8000 dilution of antisera specific for chick L-FABP ( Collins and Hargis, 1989) followed by incubation, with second antibody conjugated with alkaline phosphatase (1:10,000 rabbit antigoat IgG). Color density determination was a modification of the procedure of Velleman (1995).
Fatty acid binding activity in the cytosol of different tissues also was determined by a modification of the procedure of Morrow and Martin (1983). Cytosolic samples from each tissue were applied to a Sephadex G-75 gel filtration column, and those fractions corresponding to the molecular weight range of FABP were collected and pooled. FABP activity was determined by a radiolabelled binding assay as described by Glatz et al. (1984) and reported as DPM/mg cytosolic protein.
The chick L-FABP antisera cross-reacted with liver cytosol preps from broiler chicks (4 weeks of age), turkey poults (6 days) and turkey embryos. In a comparison with protein standards, turkey liver FABP has an approximate molecular weight of 14 kD. The concentration of L-FABP increased throughout embryonic development and after hatch. The observed increases during the later stages of incubation correlate well with documented increases in liver lipid and the movement of fatty acids between the yolk and the embryo (Ding et al., 1995; Ding and Lilburn, 1996). The increased concentration from hatch through 6 days posthatch may be reflective of the major physiologic fluxes that occur in liver lipid metabolism during this age period.
The chick L-FABP antisera cross-reacted with intestinal FABP and allowed for immunoblot quantitation of I-FABP. Both the immunoblot and ligand binding assay showed that prior to hatch (28 days), there is little FABP in the intestine. FABP specific activity (ng/mg protein) increased approximately 40% between hatch and 3 days of age, but total intestinal activity (ng/intestine) increased 10-fold. This is most likely a response to the presence of dietary fatty acids and the involvement of FABP in fatty acid absorptive processes.
A major thrust of our research is to try to understand better the mechanisms involved in embryonic transfer from the yolk to the embryo. It was interesting that there was no cross-reactivity between chick L-FABP antisera and yolk sac membrane cytosol. The ligand binding assay, however, showed that FABP specific activity (DPM/mg protein) peaked between 16 and 19 days of incubation, whereas total yolk sac membrane activity was high between 19 days and hatch (day 28). This is the period during which yolk lipids are extensively mobilized and transferred from the yolk to the developing embryo (Ding and Lilburn, 1996). The absence of any cross-reactivity between chick L-FABP antisera and YS-FABP suggested that there was only one iso-form of the protein present in yolk sac membrane.
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