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N M Bass

Publications and source records attributed to N M Bass.

At least 73 records · Page 4Linked to original sources
PubMed1991

Hepatic transport of a fluorescent stearate derivative: electrochemical driving forces in intact rat liver.

We determined the effect of varying the transmembrane Na+ electrochemical gradient on extraction of a fluorescent derivative of stearate, 12-N-methyl-7-nitrobenzo-2-oxa-1,3,-diazol-amino stearate (NBD-stearate), by the isolated perfused rat liver. Membrane potential difference (PD) of individual hepatocytes and extraction of NBD-stearate were measured simultaneously under basal conditions and during changes in PD induced by perfusate ion substitutions. Under basal conditions, PD average -30 +/- 1 mV, and extraction of 10 microM NBD-stearate from 1% albumin solutions averaged 0.54 +/- 0.03. Fluorescence microscopy indicated that uptake exhibited a declining portal-to-central gradient in the presence but not absence of Na+. Substitution of nitrate for Cl- hyperpolarized PD to -59 mV and increased extraction to 131% of control values. Withdrawal of nitrate and substitution of gluconate for Cl- depolarized PD to -3 and -15 mV, respectively, and decreased extraction to 63 and 73% of control values. Substitution of choline for Na+ eliminated the out-to-in Na+ gradient, depolarized PD to -16 mV, and decreased extraction to 27% of control values, an effect greater than expected for membrane depolarization alone. Uptake of NBD-stearate was saturable and caused Na(+)-dependent membrane depolarization at higher concentrations (300 microM). These studies indicate that uptake of NBD-stearate occurs in large part by an efficient Na(+)-dependent mechanism compatible with electrogenic Na(+)-fatty acid cotransport.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Fatty acid binding sites of rodent adipocyte and heart fatty acid binding proteins: characterization using fluorescent fatty acids.

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)

Adipose Tissue↗
PubMed1990

Functions of fatty acid binding proteins.

Cytosolic fatty acid binding proteins (FABP) belong to a gene family of which eight members have been conclusively identified. These 14-15 kDa proteins are abundantly expressed in a highly tissue-specific manner. Although the functions of the cytosolic FABP are not clearly established, they appear to enhance the transfer of long-chain fatty acids between artificial and native lipid membranes, and also to have a stimulatory effect on a number of enzymes of fatty acid metabolism in vitro. These findings, as well as the tissue expression, ligand binding properties, ontogeny and regulation of these proteins provide a considerable body of indirect evidence supporting a broad role for the FABP in the intracellular transport and metabolism of long-chain fatty acids. The available data also support the existence of structure- and tissue-specific specialization of function among different members of the FABP gene family. Moreover, FABP may also have a possible role in the modulation of cell growth and proliferation, possibly by virtue of their affinity for ligands such as prostaglandins, leukotrienes and fatty acids, which are known to influence cell growth activity. FABP structurally unrelated to the cytosolic gene family have also been identified in the plasma membranes of several tissues (FABPpm). These proteins have not been fully characterized to date, but strong evidence suggest that they function in the transport of long-chain fatty acids across the plasma membrane.

Animals↗
PubMed1990

Transfer of fluorescent fatty acids from liver and heart fatty acid-binding proteins to model membranes.

Fatty acid binding proteins (FABP) are a family of 14-15 kDa proteins found in high abundance in many mammalian cell types. The physiological functions of the FABP remain unknown. It is also not known whether each FABP has a unique function, or whether all FABP function in a similar manner in their respective tissues. In this report the rate of transfer of anthroyloxy-labeled free fatty acid (ffa) from FABP to phospholipid bilayers is monitored using a fluorescence resonance energy transfer assay. A comparison is made between heart muscle FABP and liver FABP, and the results show that the rate of ffa transfer from the heart protein is an order of magnitude greater than the rate of transfer from the liver protein. Ffa transfer rates from both liver and heart FABP are independent of acceptor concentration and composition, suggesting that, at least in the case of model membrane acceptor vesicles, the mechanism of transfer is via aqueous diffusion rather than via collision of FABP with membranes. Since the rate of ffa transfer is likely to be important to cellular ffa traffic, these studies suggest that heart FABP may function differently within the myocyte than does liver FABP within the hepatocyte.

Animals↗
PubMed1990

Induction of fatty acid binding protein by peroxisome proliferators in primary hepatocyte cultures and its relationship to the induction of peroxisomal beta-oxidation.

The induction of liver fatty acid binding protein (L-FABP) by the peroxisome proliferators bezafibrate and clofibrate was compared with the induction of peroxisomal (cyanide-insensitive) palmitoyl-CoA oxidation in cultured rat hepatocytes maintained on a substratum of laminin-rich (EHS) gel. This substratum was chosen because marked induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was effected by bezafibrate in hepatocytes supported on EHS gel, whereas only peroxisomal palmitoyl-CoA oxidation was induced in hepatocytes maintained on collagen-coated plates. In control cells on EHS, activity of peroxisomal palmitoyl-CoA oxidation remained stable, while L-FABP abundance declined with time, and L-FABP mRNA was undetectable after 5 days. In cultures exposed to bezafibrate or clofibrate, peroxisomal palmitoyl-CoA oxidation activity was induced earlier and more rapidly than L-FABP. When fibrates were withdrawn, peroxisomal palmitoyl-CoA oxidation declined rapidly, whereas L-FABP continued to increase. L-FABP induction was accompanied by a striking increase in mRNA specifying this protein. Tetradecylglycidic acid, an inhibitor of carnitine palmitoyltransferase I, effectively doubled peroxisomal palmitoyl-CoA oxidation activity. However, tetradecylglycidic acid markedly inhibited fibrate induction of L-FABP and peroxisomal palmitoyl-CoA oxidation but, unexpectedly, did not prevent the fibrate-induced proliferation of peroxisomes. Maximal induction of both L-FABP and peroxisomal palmitoyl-CoA oxidation was produced at a bezafibrate concentration in the culture medium (0.05 mM) much lower than that of clofibrate (0.3 mM). Also, bezafibrate, but not clofibrate, inhibited [1-14C]oleic acid binding to L-FABP with a Ki = 9.5 microM. We conclude that hepatocytes maintained on EHS gel provide an important tool for investigating the regulation of L-FABP. These studies show that the induction of peroxisomal beta-oxidation and L-FABP by peroxisome proliferators are temporally consecutive but closely related processes which may be dependent on a mechanism distinct from that which leads to peroxisome proliferation. Furthermore, the mechanism of action of the more potent peroxisome proliferator, bezafibrate, may be mediated, in part, by interaction of this agent with L-FABP.

Animals↗
PubMed1990

Metabolism of 15-hydroxyeicosatetraenoic acid by Caco-2 cells.

Monolayers of Caco-2 cells, a human enterocyte cell line, were incubated with [1-14C]15-hydroxyeicosatetraenoic acid (15-HETE), a lipid mediator of inflammation, and [1-14C]arachidonic acid. Both fatty acids were taken up readily and metabolized by Caco-2 cells. [1-14C]Arachidonic acid was directly esterified in cellular phospholipids and, to a lesser extent, in triglycerides. When [1-14C]15-hydroxyeicosatetraenoic acid was incubated with Caco-2 cells, about 10% was directly esterified into cellular lipids but most (55%) was beta-oxidized to ketone bodies, CO2, and acetate, with very little accumulation of shorter carbon chain products of partial beta-oxidation. The radiolabeled acetate generated from beta-oxidation of [1-14C]15-hydroxyeicosatetraenoic acid was incorporated into the synthesis of new fatty acids, primarily [14C]palmitate, which in turn was esterified into cellular phospholipids, with lesser amounts in triglycerides. Caco-2 cells were also incubated with [5,6,8,9,11,12,14,15-3H]15-hydroxyeicosatetraenoic acid; most of the radiolabel was recovered either in ketone bodies or in [3H]palmitate esterified in phospholipids and triglycerides, demonstrating that most of the [3H]15-hydroxyeicosatetraenoic acid underwent several cycles of beta-oxidation. The binding of both 15-hydroxyeicosatetraenoic acid and arachidonic acid to hepatic fatty acid binding protein, the only fatty acid binding protein in Caco-2 cells, was measured. The Kd (6.0 microM) for 15-HETE was three-fold higher than that for arachidonate (2.1 microM).

Arachidonic Acid↗
PubMed1989

Studies of the fatty acid-binding site of rat liver fatty acid-binding protein using fluorescent fatty acids.

Rat liver fatty acid-binding protein (FABP) is a 14.3-kDa cytosolic protein which binds long chain free fatty acids (ffa) and is believed to participate in intracellular movement and/or distribution of ffa. In the studies described here fluorescently labeled ffa were used to examine the physical nature of the ffa-binding site on FABP. The fluorescent analogues were 16- and 18-carbon ffa with an anthracene moiety covalently attached at eight different points along the length of the hydrocarbon chain (AOffa). Emission maxima of all FABP-bound AOffa were found to be considerably blue-shifted with respect to emission of phospholipid membrane-bound AOffa, suggesting a high degree of motional constraint for protein-bound ffa. Large fluorescence quantum yields and long excited state life-times indicate that the FABP-binding site for ffa is highly hydrophobic. Analysis of rotational correlation times for the FABP-bound AOffa suggest that the ffa are tightly bound to the protein. Variation of the quantum yield with attachment site suggests that the carboxylic acid group of the fatty acyl chain is located near the aqueous surface of the FABP. The rest of the ffa hydrocarbon chain is buried within the protein in a hydrophobic pocket and is particularly constrained at the midportion of the acyl chain.

Animals↗
PubMed1989

Acinar heterogeneity of fatty acid binding protein expression in the livers of male, female and clofibrate-treated rats.

Liver fatty acid binding protein may play a role in the intracellular transport and compartmentation of long-chain fatty acid metabolism. The distribution of liver fatty acid binding protein in the hepatic acinus was determined by means of immunocytochemistry as well as by measurement of liver fatty acid binding protein in cellular protein selectively released from zone 1 and zone 3 cells by means of anterograde and retrograde liver perfusion with digitonin. In untreated male rats, specific immunocytochemical staining for liver fatty acid binding protein showed a declining portal-to-central hepatocellular gradient in intensity, consistent with the portal-to-central ratio of liver fatty acid binding protein abundance measured in effluents from digitonin-perfused livers of 1.6:1. Female and clofibrate-treated male rats, in both of which hepatic synthesis and abundance of liver fatty acid binding protein are greater than in untreated males, differed as well in the pattern of acinar expression of this protein. In females, periportal concentrations of liver fatty acid binding protein determined from the effluent of livers perfused anterograde with digitonin were similar to male values, whereas liver fatty acid binding protein concentration in pericentral hepatocytes determined from the effluent of retrograde perfused livers was increased, resulting in a marked attenuation of the portal-to-central gradient of this protein; this was also apparent on immunocytochemistry. Clofibrate-treated rats, in contrast, displayed a panacinar increase in liver fatty acid binding protein with maintenance of the portal-to-central ratio observed in untreated males. We conclude that there exists a declining portal-to-central gradient in liver fatty acid binding protein cellular abundance in the hepatic acinus of untreated male rats. Furthermore, the increased synthesis and abundance of liver fatty acid binding protein in female and clofibrate-treated male rats results in two different alterations in the acinar expression of this protein, i.e. a pericentral increase (female) or a panlobular increase (clofibrate). Elucidation of the relationship between the zonation of hepatic fatty acid metabolism and the acinar expression of liver fatty acid binding protein should provide a more detailed understanding of the function of this protein.

Alanine Transaminase↗
PubMed1989

The hepatocellular transport of sulfobromophthalein-glutathione by clofibrate treated, perfused rat liver.

The hypolipidemic drug clofibrate is known to affect the hepatic transport of various organic anions including bilirubin, fatty acids and sulfobromophthalein. Changes in the rate of metabolism and/or intracellular transport have been claimed responsible for the effect. To evaluate these possibilities, the transport of sulfobromophthalein-glutathione, a model compound that does not require metabolism for biliary excretion, was studied in perfused livers isolated from clofibrate-treated and control rats. Cytosolic fatty acid binding protein and glutathione S-transferase activity were also measured. Clofibrate treatment significantly increased liver weight; as a result glutathione S-transferase activity (toward 1-chloro-2,4-dinitrobenzene) fell if expressed per gram of liver (4560 +/- 420 (SE) vs 7010 +/- 260 nmoles/min for clofibrate treated and controls respectively, p less than 0.002), but was unchanged when expressed per total liver (60.8 +/- 6.5 vs 64.6 +/- 3.5 mumoles/min for clofibrate and controls p greater than 0.5). Irrespective of how it was expressed fatty acid binding protein was significantly increased by the drug treatment. Steady state sulfobromophthalein-glutathione removal velocity was saturable with increasing concentrations of sulfobromophthalein-glutathione in both control and clofibrate-treated livers. Steady state extraction ratio, as well as Vmax and Km for removal, did not differ between the two groups. In keeping with other observations, these data collectively indicate that the hepatic steady state removal of nonmetabolized compounds is not affected by clofibrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Increased sinusoidal volume and solute extraction during retrograde liver perfusion.

Retrograde isolated liver perfusion has been used to probe acinar functional heterogeneity, but the hemodynamic effects of backward flow have not been characterized. In this study, extraction of a long-chain fatty acid derivative, 12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazol-amino stearate (12-NBDS), was greater during retrograde than during anterograde perfusion of isolated rat liver. To determine whether hemodynamic differences between anterograde and retrograde perfused livers could account for this finding, the hepatic extracellular space was measured for both directions of flow by means of [14C]sucrose washout during perfusion as well as by direct measurement of [14C]sucrose entrapped during perfusion. A three- to fourfold enlargement of the total hepatic extracellular space was found during retrograde perfusion by both approaches. Examination of perfusion-fixed livers by light microscopy and morphometry revealed that marked distension of the sinusoids occurred during retrograde perfusion and that this accounts for the observed increase in the [14C]sucrose space. These findings support the hypothesis that maximum resistance to perfusate flow in the isolated perfused rat liver is located at the presinusoidal level. In addition, increased transit time of perfusate through the liver and greater sinusoidal surface area resulting from sinusoidal distension may account for the higher extraction of 12-NBDS and possibly other compounds by retrograde perfused liver.

4-Chloro-7-nitrobenzofurazan↗
PubMed1988

Patent foramen ovale in young stroke patients.

The prevalence of patent foramen ovale in patients presenting with non-haemorrhagic stroke or transient ischaemic attacks under the age of 40 years was determined by contrast echocardiography. Studies were performed at rest and with a Valsalva manoeuvre in 40 stroke patients and in an age and sex matched control group. Right-to-left shunting was found in 20 (50%) of the stroke patients and 6 (15%) of the controls (p less than 0.001). Paradoxical embolism through a patent foramen ovale may be an under-recognised cause of stroke in young adults.

Acute Disease↗
PubMed1988

Isolation and characterization of fatty acid binding proteins from mammary tissue of lactating rats.

A protein fraction with fatty acid binding activity has been isolated from mammary tissue from lactating rats by a process involving DEAE-cellulose ion-exchange chromatography, heat treatment, CM-cellulose ion-exchange chromatography and finally ammonium sulphate precipitation. The purified fraction migrated as a single band on SDS/polyacrylamide-gel electrophoresis with an apparent molecular mass of 14400. However, when this protein fraction was electrophoresed under non-dissociating conditions, two species were observed in a 4:1 ratio. The two components were separated using h.p.l.c. Both bind fatty acids and appear to have similar amino acid compositions although exhibiting different pI values of 4.8 and 4.9. The mammary fatty acid binding proteins appear to be very similar to the fatty acid binding protein isolated from rat heart based on the electrophoretic mobilities and amino acid composition. The major mammary form (pI 4.9) has been partially sequenced and the amino acid sequences obtained can be aligned with 67 residues of the revised rat heart amino acid sequence [Heuckeroth, Birkenmeier, Levin & Gordon (1987) J. Biol. Chem. 262, 9709-9717]. Both mammary species also showed immunochemical identity to rat heart fatty acid binding protein when tested with an anti-serum raised against the heart protein. Anti-sera raised against the minor mammary form (pI 4.8) specifically precipitated this form under non-denaturing conditions but both forms after they had been denatured. Quantitative immunoassays using the anti-(heart fatty acid binding protein) serum showed that concentrations of the fatty acid binding proteins present in mammary cytosols increase during lactation and increase further after feeding a high-fat diet.

Amino Acid Sequence↗
PubMed1988

Revision of the blocked N terminus of rat heart fatty acid-binding protein by liquid secondary ion mass spectrometry.

The primary structure of rat heart muscle fatty acid-binding protein was investigated by liquid secondary ion mass spectrometry. The protein was digested with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease and the resulting peptides were separated by reverse phase high performance liquid chromatography. The masses of the protonated molecular ions (MH+) of the tryptic, chymotryptic, and S. aureus protease peptides were determined by liquid secondary ion mass spectrometry analysis using 20-500 pmol of material. From the tryptic digest, two peptides with MH+ 1036 and 861 were initially found that did not match the published primary sequence (Sacchettini, J. C., Meininger, T. A., Lowe, J. B., Gordon, J. I., and Banaszak, L. J. (1987) J. Biol. Chem. 262, 5428-5430). The amino acid sequences of these two peptides were determined by a combination of mass spectrometry, B/E-linked scanning, and high performance tandem mass spectrometric techniques to be: (Formula: see text). These new data require that corrections be made to the previously published sequence, involving residues 1-4 and 51-52. The corrected amino sequence for rat m-FABP reveals greater homology with myelin P2, mouse adipocyte p422 protein, and intestinal fatty acid-binding protein than was previously demonstrated.

Amino Acid Sequence↗