Interaction of nucleotides with acidic fibroblast growth factor (FGF-1).

A wide variety of nucleotides are shown to bind to acidic fibroblast growth factor (aFGF) as demonstrated by their ability to (1) inhibit the heat-induced aggregation of the protein, (2) enhance the thermal stability of aFGF as monitored by both intrinsic fluorescence and CD, (3) interact with fluorescent nucleotides and displace a bound polysulfated naphthylurea compound, suramin, (4) reduce the size of heparin-aFGF complexes, and (5) protect a reactive aFGF thiol group. The binding of mononucleotides, diadenosine compounds (ApnA), and inorganic polyphosphates to aFGF is enhanced as the degree of phosphorylation of these anions is increased with the presence of the base reducing the apparent binding affinity. The nature of the base appears to have much less effect. Photoactivatable nucleotides (8N3-ATP, 2N3-ATP, 8N3-GTP, and 8N3-Ap4A) were employed to covalently label the aFGF nucleotide binding site. In general, Kd's in the low micromolar range are observed. Protection against 90% displacement is observed at several hundred micromolar nucleotide concentration. Using 8N3-ATP as a prototypic reagent, photolabeled aFGF was proteolyzed with trypsin and chymotrypsin and labeled peptides were isolated and sequenced resulting in the identification of 10 possible labeled amino acids (Y8, G20, H21, T61, K112, K113, S116, R119, R122, H124). On the basis of the crystal structure of bovine aFGF, eight of the prospective labeled sites appear to be dispersed around the perimeter of the growth factor's presumptive polyanion binding site. On residue (T61) is more distally located but still proximate to several positively charged residues, and another (Y8) is not locatable in crystal structures. Using heparin affinity chromatography, at least three distinct photolabeled aFGF species were resolved. These labeled complexes display diminished affinity for heparin and a reduced ability to stimulate mitogenesis even in the presence of polyanions such as heparin. In conclusion, nucleotides bind apparently nonspecifically to the polyanion binding site of aFGF but nevertheless are capable of modulating the protein's activity. Evidence for the presence of a second or more extended polyanion binding site and the potential biological significance of these results in terms of potential natural ligands of aFGF are also discussed but not resolved.

Lipid composition and membrane fluidity in the small intestine of the developing rabbit.

Characteristics of small intestinal microvillus membrane lipid composition and associated lipid fluidity were studied throughout postnatal development in the rabbit. Microvillus membranes from jejunal and ileal epithelia were isolated in high purity from 14 days to adulthood. Significant jejunal-ileal differences in microvillus membrane cholesterol to phospholipid molar ratios were observed at all ages. Despite a relatively constant cholesterol content (as percentage of total lipids) both regionally and at all ages, marked increases in the microvillus membrane cholesterol to phospholipid ratio were observed after weaning in both jejunum (14 days = 0.56, adult = 0.83) and ileum (14 days = 0.66, adult = 0.98). Further age-related changes in microvillus membrane lipid composition were characterized by a significant reduction in the weight ratio of saturated to unsaturated fatty acids in jejunum and ileum and by the emergence, in the mature animal, of a jejunal-ileal difference in total lipid content (jejunum = 1.21 mg/mg protein, ileum = 1.01 mg/mg protein). Changes in microvillus membrane lipid fluidity estimated by fluorescence polarization spectrometry directly paralleled changes in the cholesterol to phospholipid molar ratio. Fluorescence anisotropy of jejunal and ileal microvillus membranes increased during the weaning period, suggesting decreased membrane fluidity. At all ages, ileal membranes were relatively less fluid than jejunal microvillus membrane. Studies of protein-free microvillus membrane liposomes, prepared from extracted membrane lipid, confirmed a major lipid-dependent thermotropic transition point at approximately 23 degrees C in adult jejunal and approximately 25 degrees C in adult ileal enterocyte membranes, although no clear transition point was observed in suckling microvillus membrane. These combined data suggested that significant alterations in the microvillus membrane lipid composition and its physical-chemical properties occur during postnatal development in both jejunal and ileal rabbit small intestinal microvillus membrane. It is proposed that membrane lipid-dependent structural characteristics may be directly related to the maturation of enterocyte transport function.