Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Fatty acid-binding protein (FABP) from bovine heart, a 15 kDa cytoplasmic protein has been investigated by multi-dimensional homonuclear and heteronuclear NMR-spectroscopy. Perdeuterated palmitic acid has been used as fatty acid ligand. The tertiary structure has been determined from distance geometry calculations with the variable target functions algorithm (DIANA) utilizing 1027 interproton distance constraints, which were obtained from 1H-homonuclear NOESY spectra. Overlapping NOE crosspeaks were assigned by heteronuclear multidimensional NMR-experiments with a 15N-labelled sample. The tertiary structure resembles a beta-barrel (beta-clam) consisting of ten anti-parallel beta-strands and a short helix-turn-helix motif. The beta-strands are arranged in two nearly orthogonal beta-sheets composed of 5 strands each. The solution structure is compared with the x-ray crystal structure of bovine heart and rat intestinal FABPs.
Mol Cell Biochem
PMID:Solution structure of bovine heart fatty acid-binding protein (H-FABPc). 823 57

Intraalveolar fibrin formation is a hallmark of many acute and chronic lung inflammatory processes. We investigated the influence of fibrin polymerization on biochemical and biophysical properties of a calf lung surfactant extract (CLSE) used for therapy of neonatal distress syndrome. Thrombin-induced coagulation of human fibrinogen (range, 0.04 to 4 mg/ml) in the presence of CLSE (2 mg/ml phospholipids) resulted in progressive loss of surface tension-lowering properties and adsorption facilities of this surfactant preparation; the CLSE-inhibitory capacity of desAABB-fibrin surpassed that of fibrinogen by more than two orders of magnitude. In parallel with the loss of surface activity, association of the predominant surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) (14C-labeled, admixed to 2 mg/ml CLSE) with polymerizing desAABB-fibrin occurred. A volume of 0.3 mg/ml insoluble fibrin effected a approximately 50% loss, and 0.6 mg/ml a > 90% loss, of DPPC from the aqueous phase. Dioleoylphosphatidylcholine, dipalmitoylphosphatidic acid, stearic acid, palmitic acid, and arachidonic acid, admixed to CLSE as labeled compounds, as well as total CLSE phospholipids were retained in polymerizing desAABB-fibrin with dose-effect curves superimposable to that of DPPC; no fibrin association was noted for 14C-glycerol-3-phosphate. Polymerizing desAA-fibrin, generated by incubation of CLSE-fibrinogen mixtures with arvin, captured DPPC and resulted in loss of surface properties at even lower concentrations, compared with desAABB-fibrin. In contrast, CLSE incubation with preformed desAABB- and desAA-fibrin polymers did not cause substantial phospholipid coupling with the clot material or loss of surface properties. Microtiter plate-immobilized fibrinogen and desAABB- and desAA-fibrinomonomers did not bind CLSE phospholipids enriched with 14C-DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1993 Aug
PMID:Lung surfactant phospholipids associate with polymerizing fibrin: loss of surface activity. 833 88

Severe deterioration of surfactant function is noted under conditions of plasma protein leakage into the alveolar space; moreover, fibrinogen has previously been reported to possess strong surfactant inhibitory capacity. Dissolution of alveolar deposits of fibrinogen and fibrin (e.g., hyaline membranes) requires enzymatic degradation by the plasminogen/plasmin system or by leukocyte-derived proteases. We investigated the surfactant inhibitory properties of differently prepared sets of fibrinogen cleavage products. Proteolysis was performed with plasmin, with predominant split products D (mol wt 85,000) and E (mol wt 50,000). In addition, fibrinogen was cleaved by leukocyte elastase and trypsin, with fragments ranging mainly between mol wt of 30,000 and 50,000. To provide split products of even lower molecular weight, fibrinogen was incubated sequentially with trypsin and endoproteinase (split products < mol wt 25,000). Natural surfactant extracts used in clinical replacement studies (CLSE, Alveofact, Curosurf, Survanta) as well as an apoprotein-based phospholipid mixture (PLM-C/B; DPPC:PG:PA = 68.5:22.5:9 with 2% [wt/wt] nonpalmitoylated recombinant human SP-C and 1% [wt/wt] natural bovine SP-B) were employed. Experiments were performed in a pulsating bubble surfactometer (standard phospholipid concentration 2 mg/ml) with assessment of surfactant activity measuring adsorption and dynamic surface tension. Fibrinogen caused dose-dependent, severe deterioration of the surface activities of Curosurf and Survanta, whereas CLSE, Alveofact, and PLM-C/B were only moderately affected up to protein-surfactant ratios of 4:1.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1993 Sep
PMID:Proteolytic cleavage of fibrinogen: amplification of its surfactant inhibitory capacity. 839 60

Expression of the beta 2-microglobulin (beta 2-m) and major histocompatibility complex (MHC) class I genes is coordinately regulated. By ligation-mediated polymerase chain reaction, we have analyzed in vivo factor binding to the promoter region of the murine beta 2-m gene. In adult spleen, in which beta 2-m is expressed, strong protection was found in three elements. Two of these elements, the beta 2-m NF-kappa B binding site and the interferon consensus sequence, are homologous to the regulatory elements of the MHC class I genes and were also found to be protected in spleen. A third protected element, PAM, identified in this work, is unique to the beta 2-m gene. None of the elements showed protection in brain tissue, in which neither the beta 2-m nor the MHC class I gene is expressed. In vivo footprinting was also performed with F9 embryonal carcinoma cells, in which expression of the beta 2-m and MHC class I genes is induced at a low level only upon stimulation with retinoic acid (RA). No in vivo protection was detected before and after RA treatment of F9 cells, indicating that RA induction of beta 2-m (and MHC class I) expression occurs without detectable in vivo factor occupancy, whereas EL4 T lymphocytes expressing beta 2-m at a high level exhibited strong protection similar to that in spleen. Despite the lack of in vivo occupancy, the nuclear factors specific for each of the three elements were present in brain tissue and F9 cells as well as in spleen tissue and EL4 cells. We show that PAM, an element identified by its in vivo protection, binds nuclear factors ranging from 40 to 50 kDa in size and is capable of enhancing transcription of a reporter in F9 and other cells. Taken together, these results indicate that in vivo factor occupancy for the beta 2-m and MHC class I promoters is coordinated and occurs through a mechanism other than mere expression of relevant factors.
Mol Cell Biol 1993 Nov
PMID:A regulatory element in the beta 2-microglobulin promoter identified by in vivo footprinting. 841 59

The exhaustive matching of the protein sequence database makes possible a broadly based study of insertions and deletions (indels) during divergent evolution. In this study, the probability of a gap in an alignment of a pair of homologous protein sequences was found to increase with the evolutionary distance measured in PAM units (number of accepted point mutations per 100 amino acid residues). A relationship between the average number of amino acid residues between indels and evolutionary distance suggests that a unit 30 to 40 amino acid residues in length remains, on average, undisrupted by indels during divergent evolution. Further, the probability of a gap was found to be inversely proportional to gap length raised to the 1.7 power. This empirical law fits closely over the entire range of gap lengths examined. Gap length distribution is largely independent of evolutionary distance. These results rule out the widely used linear gap penalty as a satisfactory formula for scoring gaps when constructing alignments. Further, the observed gap length distribution can be explained by a simple model of selective pressures governing the acceptance of indels during divergent evolution. Finally, this model provides theoretical support for using indels as part of "parsing algorithms", important in the de novo prediction of the folded structure of proteins from the sequence data.
J Mol Biol 1993 Feb 20
PMID:Empirical and structural models for insertions and deletions in the divergent evolution of proteins. 844 36

Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5'-azido-salicylamido)-undecanoic acid (5' ASU) and its acetyl ester (Ac5' ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5' ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34 x 10(-7) M, evidenced a slightly higher affinity than that reported for C16-C20 fatty acids. Consistent with the binding curve, 5' ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 microM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of 125I-5' ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5' ASU and Ac5' ASU respectively. In turn, irradiation of L-FABP incubated with 5' ASU or Ac5' ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1993 Mar 10
PMID:Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5'-azido-salicylamido)-undecanoic acid derivatives. 845

Abnormal myocardial long-chain fatty acid uptake is suspected of being involved in certain types of heart disease, but the mechanism by which the heart takes up long-chain fatty acids remains unclear. The sulfo-N-succinimidyl derivatives of long-chain fatty acids have been reported to undergo covalent binding to a membrane protein and to irreversibly inhibit the transport of long-chain fatty acids by rat adipocytes (Harmon et al., 1991). It has been suggested that the membrane protein bound by these derivatives is a candidate transporter for long-chain fatty acids in adipocytes. However, myocardial membrane long-chain fatty acid-binding proteins have not yet been fully investigated. Rat hearts were isolated and perfused with a sulfo-N-succinimidyl derivative of tritium-labeled palmitate ([3H]SSP). Then the [3H]SSP-binding protein was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography and histological autoradiography. Myocardial palmitic acid uptake was examined after pretreatment of isolated perfused rat hearts with SSP. The SSP-binding protein was isolated from bovine hearts by successive chromatography, and the amino acid sequences of lysylendopeptidase-digested peptide fragments were determined. SDS-PAGE autoradiography revealed that [3H]SSP bound to an 85-90 kDa protein derived from the myocardial microsomal fraction, and histological autoradiography demonstrated that [3H]SSP radioactivity was localized to the myocardial cell membrane. Pre-incubation with SSP inhibited palmitic acid uptake by isolated perfused rat hearts. A [3H]SSP-binding protein was also found in canine and bovine hearts, and was isolated from the bovine cardiac membrane fraction. Amino acid sequencing revealed that four peptide fragments showed strong sequence homology with rat adipocyte membrane protein, which is implicated in the binding or transport of long-chain fatty acids (Abumrad et al., 1993). We conclude that the SSP-binding protein is localized to the myocardial cell membrane and might be involved in the uptake or transport of long-chain fatty acids.
J Mol Cell Cardiol 1995 Aug
PMID:Isolation of myocardial membrane long-chain fatty acid-binding protein: homology with a rat membrane protein implicated in the binding or transport of long-chain fatty acids. 852 24

Sequences of 1,862 chromosomally encoded Escherichia coli K12 proteins were examined to identify genes likely to have arisen by duplication of genes in an ancestral chromosome. The criteria for sequence relatedness were an alignment of at least 100 amino acid residues and a PAM distance (number of accepted point mutations per 100 residues separating two sequences) below 250. A total of 971 of the 1,862 proteins examined were found in 2,329 sequence-related pairs that met these criteria. Most proteins of the sequence-related pairs were related in cellular function, as judged by biochemical and/or physiological features. Many of the pairs of proteins could be grouped into sequence-related families. If such groupings were generated from ancestral genes by duplication and divergence events, through these sequence comparisons we can identify putative ancestral sequences of the present-day genes of E. coli and other organisms. The results suggest that the 971 paralogous genes could have been derived from only 204 ancestral genes. We have also shown that the process of duplication and divergence is not the exclusive mechanism of evolution of all E. coli genes. Indeed, the relationships among the sequences of multiple (in the sense of redundant) enzymes indicate that nearly half could have arisen either by convergent evolution or by lateral transfer. Therefore, not all functionally related genes need arise by duplication and divergence.
Mol Biol Evol 1995 Nov
PMID:Gene products of Escherichia coli: sequence comparisons and common ancestries. 852 50

Palmitoylation can regulate both the affinity for membranes and the biological activity of proteins. To study the importance of the palmitoylation of the Src-like tyrosine protein kinase p56lck in the function of the protein, Cys-3, Cys-5, or both were mutated to serine, and the mutant proteins were expressed stably in fibroblasts and T cells. Both Cys-3 and Cys-5 were apparent sites of palmitoylation in Lck expressed in fibroblasts, as only the simultaneous mutation of both Cys-3 and Cys-5 caused a large reduction in the incorporation of [3H]palmitic acid. The double mutant S3/5Lck was no longer membrane bound when examined by either immunofluorescence or cell fractionation. This indicated that palmitoylation was required for association of Lck with the plasma membrane. Since the S3/5Lck protein was myristoylated, myristoylation of Lck is not sufficient for membrane binding. When Cys-3, Cys-5, or both Cys-3 and Cys-5 were changed to serine in activated F505Lck, palmitoylation of either Cys-3 or Cys-5 was found to be necessary and sufficient for the transformation of fibroblasts and for the induction of spontaneous, antigen-independent interleukin-2 production in the T-helper cell line DO-11.10. Nonpalmitoylated F505Lck exhibited little activity in vivo, where it did not induce elevated levels of tyrosine phosphorylation, and in vitro, where it was unable to phosphorylate angiotensin in an in vitro kinase assay. These findings suggest that F505Lck must be anchored stably to membranes to become activated. Because palmitoylation is dynamic, it may be involved in regulating the cellular localization of p56(lck), and consequently its activity, by altering the proximity of p56(lck) to its activators and/or targets.
Mol Cell Biol 1995 Dec
PMID:Palmitoylation of either Cys-3 or Cys-5 is required for the biological activity of the Lck tyrosine protein kinase. 852 58

We compared bile formation, and biliary and liver plasma membrane composition in guinea-pigs and rats in an attempt to explain the observation that the bile flow rate and the bile acid independent fraction of bile flow (BAIF) in guinea-pigs is about five to seven times higher than in rats. Analysis of electrolytes in bile showed that bicarbonate was significantly [acid] higher in guinea-pigs while Cl-, phosphate and Ca2+ were markedly lower than in rats. High bile independent secretion in guinea-pigs was associated with a significantly lower concentration of total bile acid, phospholipid and cholesterol than in rats. Bile acid distribution studies showed that glycine conjugated chenodeoxycholate and ketolithocholate were the main bile acids in guinea-pigs, while taurine conjugated cholate and muricholate were the predominant bile acids in rats. Total fatty acid analysis of bile indicated that in rats the major fatty acids were palmitic acid (C16:0) and linoleic acid (C18:2, n-6). In guinea-pigs, the contribution of these fatty acids was lower than in rats and compensated with a significantly higher percentage of oleic acid (C18:1, n-9). Concentrations of anionic polypeptide fraction (APF), an acidic calcium binding apoprotein closely associated with biliary phospholipid and cholesterol secretion was also significantly lower in guinea-pigs. Canalicular plasma membrane analysis showed that as compared with rats, specific activities of Na+,K+ ATPase, and cholesterol and phospholipid content were markedly lower in guinea-pigs.(ABSTRACT TRUNCATED AT 250 WORDS)
Comp Biochem Physiol B Biochem Mol Biol 1995 Aug
PMID:Bile formation and hepatic plasma membrane composition in guinea-pigs and rats. 857 19


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