P D Berk

Identification, isolation, and partial characterization of a fatty acid binding protein from rat jejunal microvillous membranes.

The mechanisms by which FFA are absorbed by the gut are unclear. To examine these processes, binding of [14C]oleate to isolated rat jejunal microvillous membranes (MVM) was studied in vitro. When [14C]oleate alone or compounded with bovine serum albumin at various molar ratios was incubated with MVM aliquots, binding was time- and temperature-dependent, inhibitable by addition of excess cold oleate, and decreased by heat denaturation or trypsin digestion of the membranes. When [14C]oleate binding to heat denatured MVM, which increased continuously as a function of the free oleate concentration and was taken as a measure of nonspecific binding, was subtracted from total binding to native MVM, a curve suggestive of saturable specific binding was observed. In contrast to fatty acids, there was no specific binding of [14C]taurocholate or [35S]sulfobromophthalein to jejunal MVM. After MVM solubilization with 1% Triton X-100, affinity chromatography over oleate-agarose and elution with 7 M urea yielded a single 40,000-mol-wt protein. This Sudan Black/periodic acid-Schiff-stain-negative protein co-chromatographed on Sephadex G-100 with [14C]oleate, [14C]palmitate, [14C]arachidonate, and [14C]linoleate, but not with the [14C]oleate ester of cholesterol, [14C]phosphatidylcholine, [14C]taurocholate, or [35S]sulfobromophthalein. A rabbit antibody to the previously reported hepatic membrane fatty acid binding protein (FABP) gave a single line of immunologic identity between the FABPs of rat jejunum and rat liver membrane. It inhibited the binding of [14C]oleate to native MVM but not heat denatured MVM, and, in immunohistochemical studies, demonstrated the presence of the FABP in the apical and lateral portions of the brush border cells of the jejunum, but not on the luminal surface of esophagus or colon. These data are compatible with the hypothesis that a specific FABP plays a role in fatty acid absorption from the gut.

Oleate uptake by cardiac myocytes is carrier mediated and involves a 40-kD plasma membrane fatty acid binding protein similar to that in liver, adipose tissue, and gut.

Uptake of [3H]oleate by canine or rat cardiac myocytes is saturable, displays the countertransport phenomenon, and is inhibited by phloretin and trypsin. Cardiac myocytes contain a basic (pI approximately 9.1) 40-kD plasma membrane fatty acid binding protein (FABPPM) analogous to those recently isolated from liver, adipose tissue, and gut, unrelated to the 12-14-kD cytosolic FABP in these same tissues. An antibody to rat liver FABPPM selectively inhibits specific uptake of [3H]oleate by rat heart myocytes at 37 degrees C, but has no influence on nonspecific [3H]oleate uptake at 4 degrees C or on specific uptake of [3H]glucose. Uptake of long-chain free fatty acids by cardiac muscle cells, liver, and adipose tissue and absorption by gut epithelial cells is a facilitated process mediated by identical or closely related plasma membrane FABPs.

At physiologic albumin/oleate concentrations oleate uptake by isolated hepatocytes, cardiac myocytes, and adipocytes is a saturable function of the unbound oleate concentration. Uptake kinetics are consistent with the conventional theory.

To reexamine the role of albumin in cellular uptake of long chain fatty acids, we measured [3H]oleate uptake by isolated hepatocytes, adipocytes, and cardiac myocytes from incubations containing oleate/albumin complexes at molar ratios from 0.01:1 to 2:1. For each ratio the uptake was studied over a wide range of albumin concentrations. In all three cell types and at any given oleate/albumin ratio, the uptake appeared saturable with increasing concentrations of oleate:albumin complexes despite the fact that the unbound oleate concentration for each molar ratio is essentially constant. However, the Km but not the Vmax of these pseudosaturation curves was influenced by substrate availability. At low albumin concentrations, uptake velocities did not correlate with unbound oleate concentrations. However, observed and expected uptake velocities coincided at albumin concentrations approaching physiologic levels and were a saturable function of the oleate/albumin ratios and the consequent unbound oleate concentrations employed. Hence, under the experimental conditions employed in this study using a variety of suspended cell types, oleate uptake kinetics were consistent with the conventional theory at physiologic concentrations of albumin.

Physicochemical and immunohistological studies of a sulfobromophthalein- and bilirubin-binding protein from rat liver plasma membranes.

Affinity chromatography over bilirubinagarose and sulfobromophthalein (BSP)-agarose was used to isolate two proteins, with high affinities for bilirubin and BSP, respectively, from Triton X-100-solubilized rat liver plasma membranes. The protein eluted from either affinity column migrated as a single band of approximately 55,000 D on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and either protein cochromatographed with both [14C]bilirubin and [35S]BSP on Sephadex G-75. On gradient gels without reduction or SDS, or on Sephadex G-150, the native BSP-binding protein had an estimated molecular mass of approximately 100,000 D. After incubation with SDS, an additional Sephadex G-150 peak of molecular mass of 56,000 D was observed. Both, the 100,000- and 56,000-D G-150 peaks cochromatographed with [35S]BSP. The native protein had an isoelectric point of 3.5, stained with periodic acid-Schiff but not Sudan black, and contained 4 mol of sialic acid per mol of protein. A rabbit antibody to the BSP-binding protein gave a line of identity with both the BSP- and bilirubin-binding antigens, and inhibited the binding of [14C]bilirubin and [35S]BSP, but not [14C]oleate or [14C]taurocholate, to rat liver plasma membranes. Immunohistochemical studies revealed the presence of the antigen on all surface domains of rat hepatocytes, but not on other cell populations from normal rat liver. It was not found in other organs. These data are compatible with the hypothesis that a specific liver cell plasma membrane protein mediates the hepatocytic sequestration of bilirubin and BSP.

Isolation and partial characterization of plasma membrane fatty acid binding proteins from myocardium and adipose tissue and their relationship to analogous proteins in liver and gut

We describe a general method for isolating a class of 40 kDa plasma membrane fatty acid binding proteins which have been identified previously only in rat liver and jejunum. Proteins extracted with 2 M salt from rat adipocyte and cardiac myocyte plasma membranes were subjected to preparative isoelectric focusing. Fractions with pIs greater than or equal to 9.0 were further purified by oleate-agarose affinity chromatography and HPLC. Each tissue yielded a single 40 kDa protein which co-chromatographed with [3H]-oleate on gel permeation HPLC, and reacted on Western blots with an antibody to the corresponding hepatic membrane protein. Related plasma membrane fatty acid binding proteins have now been isolated from each of the major sites of fatty acid transport.

Studies of the hepatocellular uptake of the hepatobiliary scintiscanning agent 99mTc-DISIDA.

Bilirubin (BR) and organic anionic dyes such as sulfobromophthalein (BSP), indocyanine green (ICG) and rose bengal (RB) enter the hepatocyte by a specific non-sodium-dependent membrane transport system. Two analogous but distinct Na+-dependent transport systems effect the uptake of conjugated bile acids such as taurocholate (TC) and free fatty acids such as oleate, respectively. The mechanism of uptake of the acetanilidoiminodiacetic acid (HIDA) class of biliary scintiscanning agents is unknown. Accordingly, rat hepatocytes were isolated by collagenase perfusion of the liver and differential centrifugation, and incubated with 99mTc-diisopropyl-HIDA (99mTc-DISIDA) alone, or in the presence of various concentrations of BSP, ICG, RB, oleate or TC, with and without bovine serum albumin (BSA). Initial uptake velocity (Vo) was determined from the initial slope of the cumulative radioactivity/time curve. In albumin free media, uptake of 99mTc-DISIDA was temperature- and pH-dependent, with maximal uptake at 37 degrees C. There was virtually no uptake of inorganic 99mTc. In incubations containing 15-1500 microM 99mTc-DISIDA, Vo was saturable, with estimated Vmax = 65 nmoles/min/10(6) hepatocytes, and Km = 1200 microM. In keeping with its weak albumin binding, 99mTc-DISIDA Vo was only minimally influenced by equimolar concentrations of BSA. 99mTc-DISIDA Vo was inhibited by BR, BSP, ICG and RB, as well as by a rabbit antibody to the rat liver plasma membrane BSP/BR binding protein. Surprisingly, Vo was also inhibited by TC and oleate, but not influenced either by ouabain or by substitution of Li+ for Na+ in the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

'Albumin-receptor' uptake kinetics do not require an intact lobular architecture and are not specific for albumin.

When freshly isolated well-stirred single cell suspensions of rat hepatocytes were incubated with 5-600 microM [3H]oleate or [35S]sulfobromophthalein (BSP) in the presence of 150 microM bovine serum albumin (BSA), uptake of both ligands increased as a linear function of the total ligand concentration in the medium. By contrast, when the same ligand concentrations were incubated as 1:1 complexes with BSA, apparent saturation of ligand uptake was observed. Analogous results were obtained in incubations employing beta-lactoglobulin instead of BSA. In none of these studies did ligand uptake velocity correlate in simple fashion with the concentration of unbound ligand in the incubation medium. These studies establish that the basis for the kinetic observations termed the albumin receptor phenomenon does not require an intact hepatic lobular architecture or space of Disse, and is not specific for albumin.

The hepatocellular uptake of free fatty acids is selectively preserved during starvation.

BACKGROUND/AIMSnThe liver loses protein during fasting. This study sought to determine if hepatic protein loss during fasting selectively preserves functions important to survival such as uptake of fatty acids, which are major energy substrates in that condition.nnnMETHODSnInitial [3H]oleate uptake and efflux rates in hepatocytes from starved (for 48 hours) and fed male rats were measured in media containing 250 mumol/L albumin at oleate/albumin ratios of 0.2:1-2:1. Uptake rates of sulfobromophthalein, taurocholate, and glucose were also determined.nnnRESULTSnInitial oleate uptake rate was saturable with respect to unbound oleate concentration. Maximum initial velocity expressed per cell number did not differ between fasted and fed animals, but measured cell volume and estimated surface area were decreased in starved vs. fed hepatocytes (921 +/- 21 vs. 1623 +/- 58 microns2, respectively; P < 0.001). Consequently, when expressed per surface area, maximum initial velocity was greater in starved cells (17 +/- 3 vs. 10 +/- 2 [pmol.min-1.micron2] x 10(-7); P < 0.02). Expressed similarly, oleate efflux was also greater from starved hepatocytes and was inhibited by an antibody to plasma membrane fatty acid binding protein (FABPpm). FABPpm concentration per unit area of plasma membrane also increased in starved hepatocytes (P < 0.05). By contrast, uptake rates of sulfobromophthalein, taurocholate, and glucose by starved hepatocytes were decreased when expressed per cell number and unchanged per unit area.nnnCONCLUSIONSnDuring fasting, the hepatocellular uptake mechanism for oleate is selectively preserved compared with those for sulfobromophthalein, taurocholate, or glucose.

Oleate uptake kinetics in the perfused rat liver are consistent with pseudofacilitation by albumin.

We measured uptake of a representative free fatty acid, oleate, by the single-pass perfused rat liver at oleate:albumin molar ratios of 0.01 to 2:1. For each ratio, uptake was studied at albumin concentrations from 50 to 600 microM. When uptake velocity was plotted as a function of the albumin concentration, the data at each ratio exhibited a pseudosaturation pattern as previously observed in isolated cells (J Clin Invest 84: 1325). At a physiologic albumin concentration of 600 microM, a plot of uptake vs. unbound oleate concentrations was best fitted by the Michaelis-Menten equation (Vmax = 235 +/- 8.8 nmol.min-1.g.liver-1; Km = 130 +/- 12 nM). As the albumin concentration was increased from 50 to 250 microM, the unbound oleate clearance, calculated by either the undistributed sinusoidal or venous equilibrium models, increased progressively, in violation of conventional pharmacokinetic theory, indicating an enhancing effect of albumin on ligand uptake at low albumin concentrations. In contrast, there was no significant difference between measures of unbound clearance at albumin concentrations of 350 and 600 microM. To explain this phenomenon, the clearance data were examined for evidence of facilitation (accelerated dissociation of ligand:albumin complexes) by the clearance ratio test (square root rule). All deviations from the predictions of conventional theory were entirely attributable to pseudofacilitation. No data required explanation by a true facilitation model.

Quantitation of Plasma-Membrane Fatty Acid-Binding Protein by Enzyme Dilution and Monoclonal-Antibody Based Immunoassay

SummaryA plasma membrane fatty acid-binding protein (h-FABPPm) has been isolated from rat hepatocytes. Analogous proteins have also been identified in adipocytes, jejunal enterocytes and cardiac myocytes, all cells with high transmembrane fluxes of fatty acids. These 43 kDa, highly basic (pl = 9.1) FABPpm s appear unrelated to the smaller, cytosolic FABPs (designated FABPs) identified previously in the same tissues. h-FABPpm appears closely related to the mitochondrial isoform of glutamic-oxaloacetic transaminase (mGOT), and both the purified protein and liver cell plasma membranes (LPM) possess GOT enzymatic activity. From their relative GOT specific activities it is estimated that h-FABPpm constitutes approximately 2% of LPM protein, or about 0.7 × 107 sites per cell. A monoclonal antibody-based competitive inhibition enzyme immunoassay (CIEIA) for h-FABPpm is described; it yields an estimate of 3.4 x 107 h-FABPpm sites per hepatocyte. Quantitated by either method, h-FABPPm appears to be a highly abundant protein constituent of LPM.