Search

The keratinocyte lipid-binding protein (KLBP) has been identified on the basis of nucleotide sequence analysis of its cloned cDNA as a new member of the intracellular lipid-binding protein (iLBP) multigene family. To characterize KLBP and determine its ligand-binding properties, its cDNA was subcloned into Escherichia coli, and the protein was overexpressed and purified to homogeneity by a combination of acid extraction, gel permeation, and ion-exchange chromatographies. Purified KLBP exhibited high-affinity binding of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulfonate (1,8-ANS), displaying an apparent dissociation constant of 390 +/- 90 nM (n = 0.74 +/- 0.2). Using an assay based upon displacement of the bound fluorophore, KLBP was found to bind long chain fatty acids most avidly; oleic acid (18:1) bound with an apparent Kd of 248 +/- 12 nM, and arachidonic acid (20:4) exhibited a dissociation constant of 318 +/- 14 nM. As the length of the fatty acid decreased, the binding affinity was reduced; myristic acid (14:0) bound with a K(d) of 1409 +/- 423 nM, but medium-chain (decanoic acid, 10:0) and short-chain (octanoic acid, 8:0) lipids were not bound at all. The protein did not bind prostaglandin E2 with any measurable affinity but did associate with eicosanoids such as 5-hydroperoxyeicosatetraenoic acid (5-HPETE; K(d) of 848 +/- 211 nM) and 15-HPETE (Kd of 463 +/- 243 nM) and to a lesser extent their hydroxy derivatives, 5-HETE and 15-HETE (Kd of 1560 +/- 115 nM and greater than 4 microM, respectively). all-trans-Retinoic acid was a weak ligand for KLBP, binding with a Kd of 3600 nM, and all-trans-retinol did not displace 1,8-ANS. Molecular modeling of the KLBP sequence upon the X-ray crystal structures of several iLBP's suggested that the side chains of one or more cysteine residues may reside within the putative ligand-binding cavity. Consistent with this, sulfhydryl titration of purified KLBP with 5,5'-dithiobis(2-nitrobenzoic acid) at pH 8.0 in the presence and absence of oleic acid revealed that at least one residue was protected from modification by the fatty acid. These results describe the first purification and characterization of the ligand-binding properties of KLBP and indicate that the protein is a fatty acid binding protein with a tertiary structure likely to be similar to other members of the iLBP multigene family.

Adipocyte lipid-binding protein is a 14.6-kDa polypeptide that is responsible for the intracellular trafficking of fatty acids. Its structure previously has been solved in the apo and holo forms complexed with stearate and oleate. To examine the binding of lipids other than those with a carboxylate headgroup, we have determined the structure of ALBP in complex with a sulfonic acid, hexadecanesulfonic acid, and compared its structure with the natural fatty acid analog, palmitate. Crystallographic refinement led to similar models, both with R-factors of about 20% and a resolution of 1.6 A. results can be compared with earlier studies on C18 fatty acids, both saturated and unsaturated. The previously refined complexes with stearate and oleate in combination with the complexes of palmitate and hexadecanesulfonic acid demonstrate specific positions for water molecules bound in the internal cavity. Many of the water-binding sites are present in both the apo form and the holo forms of the protein. With ligand present, a network of 10 internalized water molecules appear to form a hydrophobic hydration region. In spite of the sp3 geometry of the sulfonic acid derivative, the headgroup occupies the same site as that of the planar carboxylate in natural fatty acids. These results demonstrate that intracellular lipid-binding proteins are capable of binding a wider variety of lipids than previously considered and reveal the importance of interior ordered water molecules in the binding cavity.

Prior methods for the chemical synthesis of phosphotyrosine-containing peptides involved the incorporation of fully protected phosphoamino acids into the peptide chain or phosphorylation of free phenol side chains after peptide assembly is complete. The present work describes a novel and general methodology for the solid-phase synthesis of phosphopeptides, featuring direct incorporation of N alpha-(9-fluorenylmethyloxycarbonyl)-O-phospho-L-tyrosine (unprotected side chain). This technique obviated the formation of peptide byproducts containing tyrosine H-phosphonate, a previously unrecognized side reaction from literature phosphorylation/oxidation approaches. Phosphopeptides corresponding to the tyrosine phosphorylation site of adipocyte lipid binding protein were synthesized by the newer, preferred method. These peptides were purified and characterized by high-performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), amino acid analysis (AAA), fast atom bombardment mass spectrometry (FABMS), and 31P nuclear magnetic resonance (31P NMR). The synthetic peptides were tested as substrates for two distinct protein tyrosine phosphatases, rat brain protein tyrosine phosphatase (PTPase) and human acid phosphatase. Substrate specificity was measured at pH 6.0 and 37 degrees C, using a colorimetric assay for released inorganic phosphate. Kinetic analysis revealed that both the rat brain PTPase and the human adipocyte acid phosphatase catalyzed peptide dephosphorylation but with different rates and affinities. The rat brain PTPase displayed classical Michaelis-Menten kinetics, with Km's of 68 +/- 9 microM and 42 +/- 11 microM and kcat/Km values of 4.9 x 10(5) s-1 M-1 and 6.9 x 10(5) s-1 M-1 determined for phosphorylated peptides of lengths 4 and 10 residues, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A substrate for the insulin receptor kinase in 3T3-L1 adipocytes has previously been identified as the adipocyte lipid-binding protein (ALBP, also known as aP2 or p15). We have characterized the effect of tyrosyl phosphorylation on ALBP structure and ligand-binding properties. Phosphorylated ALBP (phospho-ALBP) was isolated by a combination of gel filtration, anion exchange chromatography, and immunoaffinity chromatography on anti-phosphotyrosine agarose. Circular dichroic spectroscopy indicated that the phosphoprotein was similar in structure to native ALBP. Phospho-ALBP exhibited a slight decrease in calculated alpha-helical content which was compensated for by an increase in beta-sheet structure. The wavelength yielding maximum tryptophan fluorescence was unaltered by phosphorylation (334 +/- 1 nm). However, the concentration of guanidine HCl yielding 50% denaturation was 1.43 M for ALBP and 0.92 M for phospho-ALBP. The delta Goapp was 3.87 and 3.25 kcal mol-1 for ALBP and phospho-ALBP, respectively, suggesting that phosphorylation destabilized the protein. To assess the binding characteristics of the phosphoprotein, a long-chain fatty acid affinity column was synthesized to which native ALBP specifically bound. In contrast, phospho-ALBP showed little or no affinity for the column. Furthermore, phosphorylation virtually abolished binding of the fluorescent fatty acid analogue 12-(9-anthroyloxy)oleic acid. Fatty acid binding activity was recovered (approximately 60%) upon dephosphorylation with protein tyrosine phosphatase. The structural studies, coupled with the crystal structure of the apoprotein, indicate that the dramatic reduction in binding affinity is likely a result of steric hindrance in the binding cavity or of electrostatic interactions of the phosphoryl group with the fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine adipocyte and rat heart fatty acid binding proteins (FABP) are closely related members of a family of cytosolic proteins which bind long-chain free fatty acids (ffa). The physical and chemical characteristics of the fatty acid binding sites of these proteins were studied using a series of fluorescent analogues of stearic acid (18:0) with an anthracene moiety covalently attached at seven different positions along the length of the hydrocarbon chain (AOffa). Previously, we used these probes to investigate the binding site of rat liver FABP (L-FABP) [Storch et al. (1989) J. Biol. Chem. 264, 8708-8713]. Here we extend those studies to adipocyte and heart FABP, two members of the FABP family which share a high degree of sequence homology with each other (62% identity) but which are less homologous with L-FABP (approximately 30%). The results show that the fluorescence emission spectra of AOffa bound to adipocyte FABP (A-FABP) are blue-shifted relative to heart FABP (H-FABP), indicating that AOffa bound to A-FABP are held in a more constrained configuration. For both proteins, constraint on the bound ffa probe is highest at the midportion of the acyl chain. Ffa are bound in a hydrophobic environment in both proteins. Excited-state lifetimes and fluorescence quantum yields suggest that the binding site of H-FABP is more hydrophobic than that of A-FABP. Nevertheless, acrylamide quenching experiments indicate that ffa bound to H-FABP are more accessible to the aqueous environment than are A-FABP-bound ffa.(ABSTRACT TRUNCATED AT 250 WORDS)

The adipocyte lipid binding protein (ALBP) is a member of a multigene family of low molecular weight proteins which stoichiometrically and saturably bind hydrophobic ligands and presumably facilitate intracellular lipid metabolism. To probe the structure-function relationship of the binding domain of ALBP, chemical modification has been employed. Modification of the two cysteinyl residues of ALBP (Cys1 and Cys117) with a variety of sulfhydryl reagents decreased the apparent affinity for oleic acid in the following order of effectiveness: methyl methanethiosulfonate much much less than p-(chloromercuri)benzenesulfonic acid less than N-ethylmaleimide (NEM) = 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Thiol titration of ALBP with DTNB in the presence of bound oleate resulted in the modification of a single cysteinyl residue. The oleate-protected cysteine was identified as Cys117 by modification with a combination of reversible (DTNB) and irreversible (NEM) sulfhydryl reagents in the presence or absence of saturating oleic acid. Cys117-NEM ALBP exhibited a large decrease in binding affinity while Cys1-NEM ALBP exhibited normal binding properties. Neither the modification of ALBP with NEM nor the addition of oleic acid had a significant effect on protein structure, as judged by circular dichroic analysis. These results suggest that Cys117 of ALBP resides in the ligand binding domain and that site-specific modification can be utilized to assess the conformational flexibility of the binding cavity.

The portal region of fatty acid binding proteins is hypothesized to function as a dynamic aperture, controlling accessibility of external ligands to the internal fatty acid binding cavity. To test this hypothesis, a triple mutant of the murine FABP4 has been developed (V32G, F57G, K58G, referred to as the portal mutant) that is predicted to constitutively enlarge the opening due to a reduction in the molecular dimensions of the side chains of key portal amino acids. The portal mutant was purified from expressing Escherichia coli, its stability was evaluated, and the thermodynamics and kinetics of ligand binding were compared to that of wild-type protein. Introduction of the three amino acid substitutions caused no significant change in the stability of the protein with a free energy of unfolding of 13.7 kJ/mol as compared to 14.0 kJ/mol for the wild-type protein. The portal mutant exhibited a modest decrease (4-fold) in ligand binding affinity using the fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) as a surrogate ligand. 1,8-ANS displacement assays revealed that the binding affinity for oleate increased from a K0.5 of 196 +/- 15 nM for the wild-type protein to 165 +/- 8 for the portal mutant, while that for arachidonate decreased from the wild type of 186 +/- 11 nM to 418 +/- 26 nM for the portal mutant. To evaluate cavity accessibility, rate of 1,8-ANS binding was assessed between the portal and wild-type protein. Using equimolar amounts of ligand and protein at 4 degrees, 1,8-ANS bound within the cavity to 95% saturation (t0.95) in 750 ms, while the mutant protein was fully modified in less than 1.4 ms. To independently evaluate cavity accessibility, modification of the sole protein cysteine residue, C117 residing within the cavity near C2-C4 of the bound ligand, was monitored using 5,5'-dithio-bis(2-nitrobenzoic acid) modification. The half time for modification (t0.5) for the wild-type protein was approximately 20 s, while that for V32G F57G K58G occurred in less than a second. As such, enlargement of the portal region of FABP4 markedly increased the accessibility of ligands to the cavity while having only modest effects on ligand affinity. Taken together, these data provide support for the portal region hypothesis and suggest dynamic fluctuations in this region regulate cavity access, but not ligand affinity or selectivity.

Adipocyte fatty acid binding protein (A-FABP) is a 15-kDa protein found in high abundance in the cytosol of adipose cells. To better understand the role of this protein in intracellular free fatty acid (ffa) transport, the mechanism of ffa transfer from A-FABP to model membranes was examined by monitoring the transfer of fluorescent anthroyloxy ffa (AOffa) to small unilamellar phospholipid vesicles, using a resonance energy transfer assay. Structural features of ffa that increase aqueous solubility, such as shorter chain length and unsaturation, did not increase the AOffa transfer rate. In addition, solution conditions that increase the aqueous solubility of ffa, such as decreasing ionic strength and increasing pH, had little effect on AOffa transfer from A-FABP to membranes. These results suggest that AOffa do not transfer through the aqueous phase. The small entropic contribution to the free energy of the transfer process provides further evidence that AOffa may not travel through the surrounding aqueous environment when transferred from A-FABP to phospholipid membranes. Finally, the rate of AOffa transfer from A-FABP was directly dependent on the concentration of the acceptor membranes. These studies suggest that AOffa transfer from A-FABP to phospholipid vesicles may occur via transient collisional interactions between the protein and membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipocyte lipid-binding protein (ALBP) is the adipocyte member of an intracellular hydrophobic ligand-binding protein family. ALBP is phosphorylated by the insulin receptor kinase upon insulin stimulation. The crystal structure of recombinant murine ALBP has been determined and refined to 2.5 A. The final R factor for the model is 0.18 with good canonical properties. Crystalline ALBP has a conformation which is essentially identical to that of intestinal fatty acid binding protein and myelin P2 protein. Although the crystal structure is of the apo- form, a cavity resembling that in other family members is present. It contains a number of bound and implied unbound water molecules and shows no large obvious portal to the external milieu. The cavity of ALBP, which by homology is the ligand-binding site, is formed by both polar and hydrophobic residues among which is tyrosine 19. Y19 is phosphorylated by the insulin receptor kinase as described in the accompanying paper [Buelt, M. K., Xu, Z., Banaszak, L. J., & Bernlohr, D. A. (1992) Biochemistry (following paper in this issue)]. By comparing ALBP with the earlier structural results on intestinal fatty acid binding protein, it is now possible to delineate conserved amino acids which help form the binding site in this family.

The inactivation of glutamine phosphoribosylpyrophosphate amidotransferase by reaction of its iron-sulfur center with O2 is believed to be a physiologically important mode of regulation of this enzyme in Bacillus subtilis cells in the stationary phase of growth. Chemical and physical changes accompanying oxidation of the purified enzyme by O2 were studied. The iron of the 4Fe-4S center was oxidized to enzyme-bound high-spin Fe3+; the S2- was oxidized to a mixture of S0 bound as thiocystine and unidentified products. The oxidant appeared to be O2, rather than peroxide, superoxide, hydroxyl radical, or singlet oxygen. Gross physical changes in the oxidized enzyme were shown by its aggregation, decreased solubility, and altered circular dichroic spectrum. Experimental variables affecting the rate of oxidative inactivation were described; the most important of these was modulation of rates of inactivation by the allosteric inhibitors AMP, ADP, GMP, GDP and by the substrate P-Rib-PP. AMP was a potent stabilizer, whose effect was antagonized by P-Rib-PP. The other nucleotides, either acting singly or acting as synergistic pairs, were destabilizers and able to antagonize stabilization by AMP. The results are discussed in terms of the regulation of the stability of amidotransferase and its degradation in vivo.

Human adipocyte lipid-binding protein (H-ALBP) was purified from normal subcutaneous adipose tissue to greater than 98% homogeneity, utilizing a combination of acid fractionation, gel filtration, covalent chromatography on activated thiol-Sepharose 4B, and anion-exchange chromatography. Human ALBP comprised about 1% of total cytosolic protein in human adipose tissue, had a relative molecular mass of about 15 kDa, and existed as a monomer in solution. The amino terminus of H-ALBP was blocked to sequencing. When a liposome ligand delivery assay was used, H-ALBP saturably bound oleic acid with about 1 mol of ligand bound per mole of protein. Additionally, H-ALBP saturably bound retinoic acid as determined by the quenching of intrinsic tryptophan fluorescence. A full-length H-ALBP cDNA has been cloned; the sequence predicts a 649-base mRNA comprised of a 62-base 5'-noncoding region containing an 18S ribosome-binding site, a single 396-base open-reading frame, and a 191-base 3'-noncoding region. Comparative sequence analysis indicated that the 132 amino acid H-ALBP is a member of a multigene family of intracellular lipid-binding proteins and contains the consensus substrate phosphorylation sequence for tyrosyl kinases.

A procedure for the rapid and efficient purification of glutamine phosphoribosylpyrophosphate amidotransferase to better than 98% homogeneity from depressed Bacillus subtilis cells is described. The molecular weight of the subunit was estimated to be about 50 000. The purified enzyme exhibits microheterogeneity on electrophoresis on highly resolving polyacrylamide gels; it is suggested that this heterogeneity results from limited proteolytic modification of the native subunit. The native enzyme exists in equilibrium among tetrameric, dimeric, and monomeric forms. The influence of enzyme concentration and the presence of substrates and allosteric inhibitors on this equilibrium are described. There is no simple correlation between allosteric inhibition and stabilization of dimeric or tetrameric states. The amino acid composition of the amidotransferase is reported; presence of a 4Fe-4S center in the enzyme was described previously. Preparation of inactive apoprotein by treatment with 1,10-phenanthroline and general characteristics of the apoprotein are presented.