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P D Berk

Publications and source records attributed to P D Berk.

At least 19 recordsLinked to original sources
PubMed1992

Adipocyte differentiation of 3T3-L1 cells involves augmented expression of a 43-kDa plasma membrane fatty acid-binding protein.

A previously described 43-kDa plasma membrane fatty acid-binding protein (FABPPM) was not observed by immunohistochemical methods in proliferating 3T3-L1 fibroblasts. However, it was detectable in plasma membranes by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated adipocytes. These observations were confirmed by extraction of plasma membrane proteins and subsequent immunoblotting. Kinetics of initial [3H]oleate uptake by both fibroblasts and adipocytes consisted of the sum of a saturable and a non-saturable component. During differentiation the saturable component increased progressively. Vmax increased from 3 to 25 to 110 pmol.s-1.mg cell protein-1 between the fibroblast, the 4-day, and 8 day adipocyte stages; Km was 24 nM in fibroblasts and approximately 55 nM in both 4- and 8-day differentiated adipocytes. By contrast, the rate constant for nonsaturable oleate influx decreased progressively from 0.026 to 0.010 ml.s-1.mg protein-1 between the fibroblast and 8 day adipocyte stages. In 8-day adipocytes saturable oleate uptake was inhibited by up to 55% by antibodies against rat liver FABPPM; these antibodies had no effect on uptake of 2-deoxyglucose or the medium chain fatty acid octanoate. They also had no effect on oleate uptake by fibroblasts. These studies support the hypothesis that FABPPM is a component of a saturable transport mechanism for long chain fatty acids.

3T3 Cells↗
PubMed1992

Characteristics of oleate binding to liver plasma membranes and its uptake by isolated hepatocytes.

To clarify mechanisms of hepatic free fatty acid uptake, [3H]oleate uptake by isolated rat hepatocytes was studied, using solutions of 150 microM bovine serum albumin at oleate:albumin molar ratios of 0.033-6.7:1. Oleate partitioning between liver plasma membranes and albumin was also studied, and used to ascertain the membrane binding function for oleate. The experimental uptake curve was complex, but could be resolved by computer fitting into a sum of two components, one a saturable and the second a linear function of the unbound oleate concentration. The saturable component comprises > 90% of total oleate uptake when the oleate:albumin molar ratio is < 2.5, but < 50% when this ratio is > 5. Membrane binding also consisted of a sum of a saturable and a linear component. By comparison of the computer-fitted uptake and binding functions, separate rate constants for the transfer into the cell of the saturably and non-saturably bound oleate were estimated to be 0.7 s-1 and 0.05 s-1, respectively. The former is compatible with a specific, protein-mediated process. It is 15-times greater than the corresponding rate constant for transfer of non-saturably bound oleate into the cell, which in turn is similar to reported rates of non-specific 'flip-flop' of fatty acids across lipid bilayers. The observed kinetics are not consistent with models in which uptake occurs principally from the albumin-bound pool of oleate, or solely from the oleate which has partitioned passively into the lipid bilayer of the plasma membrane.

Animals↗
PubMed1992

Sex differences in hepatic fatty acid uptake reflect a greater affinity of the transport system in females.

In this study, we examined the hypothesis that the reported sex difference in hepatic free fatty acid (FFA) uptake involves the putative FFA transport system, the plasma membrane fatty acid binding protein (FABPpm). In hepatocytes isolated from both male and female rats, initial [3H]oleate uptake velocity reflected transmembrane influx and not subsequent metabolism and was a saturable function of the unbound oleate concentration. Although Vmax values were similar (61 +/- 2 vs. 65 +/- 5 pmol.min-1.5 x 10(4) cells-1 for females and males, respectively), the apparent Km was significantly smaller in females (40 +/- 4 vs. 90 +/- 11 nM; P less than 0.05), reflecting faster influx velocities in female cells over a range of unbound oleate concentrations. The oleate efflux rate constant was also greater in females (0.280 +/- 0.014 vs. 0.198 +/- 0.020 min-1; P less than 0.05) despite their greater hepatic content of cytosolic FABP. Finally, despite the greater rates of transmembrane FFA flux in female hepatocytes, the surface expression of FABPpm was virtually identical in the two sexes (2.5 +/- 0.5 vs. 2.4 +/- 0.4 microgram/10(6) cells). Collectively, these data indicate that at FFA-to-albumin ratios occurring in vivo the plasma membrane of female hepatocytes transports oleate bidirectionally at a greater rate than that of male hepatocytes. A sex-related difference in the functional affinity of FABPpm for FFA appears the most likely explanation for the greater oleate uptake in females.

Animals↗
PubMed1991

Hepatocellular 22Na+ uptake: effect of oleate.

Whether cellular oleate uptake is Na+ coupled remains controversial. Our present studies document that hepatocellular [3H]oleate uptake is unaltered by isomotic substitution of Na+ with K+, Li+, or sucrose in Hanks' HEPES buffer. In parallel studies 22Na+ uptake was significantly (P less than 0.02) increased by concentrations of alanine that yielded [3H]-alanine uptakes greater than 24% of basal 22Na+ uptake when both were expressed as nanomoles per minute per 10(6) hepatocytes. Although [3H]glutamine uptake exceeded this threshold, maximal specific [3H]taurocholate uptake did not. Consistent with the observations with alanine, addition of glutamine, but not taurocholate, to the incubations resulted in a significant increase in 22Na+ uptake. The ionophore monensin increased uptake of 22Na+ under all conditions. Both in the absence and in the presence of HCO3- (4 and 25 mM), specific [3H]oleate uptake was sufficient to elicit a readily detectable effect on 22Na+ uptake, if Na+ and oleate were cotransported. However, addition of 1 mM oleate did not affect 22Na+ uptake in the absence of HCO3- (5.4 +/- 0.6 vs. control 5.4 +/- 0.4 nmol.min-1.10(6) cells-1) as well as in the presence of 4mM HCO3- (10.4 +/- 1.8 vs. control 9.9 +/- 1.1) and 25 mM HCO3- (10 +/- 1.3 vs. control 10.8 +/- 0.5). These data indicate that the predominant component of hepatocellular oleate uptake is not directly associated with Na+ influx. They do not exclude, however, a more complex or indirect link.

Alanine↗
PubMed1991

Phenobarbital does not increase early labeling of bilirubin from 4-[14C]-delta-aminolevulinic acid in man and rat.

delta-Aminolevulinic acid-4-[14C] and [3H]-bilirubin were administered intravenously to five patients with Gilbert's syndrome and four healthy control subjects on two occasions: before and on days 10 through 14 of a course of phenobarbital (2.5 mg/kg/day). The resulting curves of [3H]-bilirubin and [14C]-bilirubin in plasma were analyzed by computer to determine a number of parameters of physiological interest. As expected, phenobarbital produced a highly significant fall in the plasma concentration of unconjugated bilirubin as a result of a significant increase in hepatic bilirubin clearance in all subjects; plasma bilirubin turnover was unaltered. Surprisingly, the drug produced no change in the incorporation of [14C]-delta-aminolevulinic acid into [14C]-early labeled bilirubin. To explain this unexpected finding, the effects of phenobarbital (75 mg/kg/day for 6 days) on incorporation of [14C]-delta-aminolevulinic acid and 2-[14C]-glycine into [14C]-early labeled bilirubin and on the activity of the enzyme delta-aminolevulinic acid synthase were studied in nonfasted, adult, male Sprague-Dawley rats. At the dose and duration of treatment used, phenobarbital administration increased total hepatic delta-aminolevulinic acid synthase activity and produced a significant increase of 70% in the incorporation of [14C]-glycine into early labeled bilirubin. By contrast, no increase in the incorporation of [14C]-delta-aminolevulinic acid into early labeled bilirubin was observed. These data suggest that delta-aminolevulinic acid is an inappropriate precursor for studies of the rate of heme biosynthesis, presumably because it bypasses delta-aminolevulinic acid synthase, the physiological rate-limiting enzyme in the heme biosynthetic pathway.

Aminolevulinic Acid↗
PubMed1990

Purification of rat liver mitochondrial aspartate aminotransferase and separation of its isoforms utilizing high-performance liquid chromatography.

A rapid method for purification of mitochondrial aspartate aminotransferase from rat liver employing high-performance liquid chromatography is reported. The product is purified 80-fold with a recovery greater than or equal to 50% in a single day. The amino acid composition, N-terminal amino acid sequence, specific activity, and spectral characteristics of the isolated enzyme are similar to those previously reported for this protein. The protein is homogeneous by standard electrophoretic and chromatographic criteria, but can be resolved into at least five isoforms by a carboxymethylated resin column using high-performance liquid chromatography. The principal isoform initially isolated is converted into two additional isoforms with lower specific activity upon storage at 4 degrees C.

Amino Acid Sequence↗
PubMed1990

Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related.

The hepatic plasma membrane fatty acid-binding protein (h-FABPPM) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate with a Ka approximately 1.2-1.4 x 10(7) M-1. Sinusoidally enriched liver plasma membranes and purified h-FABPPM have GOT enzymatic activity; the relative specific activities (units/mg) of the membranes and purified protein suggest that h-FABPPM constitutes 1-2% of plasma membrane protein in the rat hepatocyte. Monospecific rabbit antiserum against h-FABPPM reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of [3H]oleate but not that of [35S]sulfobromophthalein or [14C]taurocholate. The inhibition of oleate uptake produced by anti-h-FABPPM can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABPPM and mGOT are closely related.

Amino Acid Sequence↗
PubMed1989

Comparison of erythroid progenitor cell growth in vitro in polycythemia vera and chronic myelogenous leukemia: only polycythemia vera has endogenous colonies.

The ability of erythroid cultures to distinguish among myeloproliferative disorders was examined. We studied 14 patients with polycythemia vera (PV), 11 with chronic myelogenous leukemia (CML), four with non-PV erythrocytosis, two with agnogenic myeloid metaplasia, as well as three normal fetuses and greater than 25 normal adults. Endogenous, i.e. grew without added erythropoietin, bone marrow CFU-E-derived colonies were observed in all but one PV patient. However, endogenous blood BFU-E-derived bursts were observed in only eight of 14 PV patients. Endogenous erythroid colonies were not seen in cultures from any normal adults or fetuses, or patients with CML, erythrocytosis, or myeloid metaplasia. In PV, relative HbF synthesis was always greater in cultures without erythropoietin, while in cultures from all other patients relative HbF synthesis was similar to that observed in cultures from normal individuals. We conclude that PV and CML are distinguishable in culture since CML patients do not have endogenous growth. Most important, endogenous bone marrow CFU-E-derived colonies are the only consistently unique observation in patients with PV, and endogenous CFU-E- and BFU-E-derived colonies and bursts are not uniformly observed in PV blood cultures. In-vitro studies of erythropoiesis to confirm the diagnosis of PV, therefore, require marrow when endogenous colonies and bursts are absent from blood cultures.

Adolescent↗