Showing papers on "Bovine serum albumin published in 1979"

Transport of Steroid Hormones Through the Rat Blood-Brain Barrier. Primary Role of Albumin-Bound Hormone

Abstract: These studies were undertaken to investigate (a) the permeability properties of the blood-brain barrier (BBB) to the major gonadal and adrenal steroid hormones, and (b) the role of the binding proteins of plasma (albumin and specific globulins) in the regulation of BBB steroid hormone transport. The permeability of the BBB to [(3)H]-labeled progesterone, testosterone, estradiol, corticosterone, aldosterone, and cortisol, was measured relative to [(14)C]butanol, a freely diffusable reference, in the barbiturate anesthetized rat using a tissue sampling-single injection technique. The isotopes were rapidly injected in a 200-mul bolus of Ringer's solution (0.1 g/dl albumin) via the common carotid artery and the percent extraction of unidirectional influx of hormone was determined after a single pass through brain: progesterone, 83+/-4%; testosterone, 85+/-1%; estradiol, 83+/-3%; corticosterone, 39+/-2%; aldosterone, 3.5+/-0.8%; and cortisol, 1.4+/-0.3%. The selective permeability of the BBB was inversely related to the number of hydrogen bonds each steroid formed in aqueous solution and directly related to the respective 1-octanol/Ringer's partition coefficient. When the bolus injection was 67% human serum, >95% of the labeled steroid was bound as determined by equilibrium dialysis. However, the influx of the steroids through the BBB was inhibited by human serum to a much less extent than would be expected if only the free (dialyzable) hormone was transported; progesterone, estradiol, testosterone, and corticosterone transport was inhibited 18, 47, 70, and 85% respectively, or in proportion to the steroid binding to plasma globulins. Rat serum (67%) only inhibited the transport of these four hormones, 0, 13, 12, and 69%, respectively, reflecting the absence of a sex hormone-binding globulin in rat plasma. However, neonatal rat serum (67%) inhibited progesterone, testosterone, and estradiol transport 0, 0, and 91%, respectively, consistent with the presence of an estradiol-binding protein in neonatal rat serum. The binding of steroid hormone to bovine albumin in vitro (as determined by equilibrium dialysis) was compared to albumin binding in vivo (as determined by the single injection technique). The ratio of apparent dissociation constant in vivo, K(D)(app), to the in vitro K(D) was: >>200 for progesterone, >200 for testosterone, 120 for estradiol, and 7.7 for corticosterone. Assuming the steady-state condition, the K(D)(app)/K(D) was found to be proportional to the BBB permeability for each steroid. These data demonstrate (a) the selective permeability properties of the BBB to the major steroid hormones is proportional to the tendency of the steroid to partition in a polar lipid phase and is inversely related to the number of hydrogen bond-forming functional groups on the steroid nucleus; (b) the presence of albumin in serum may bind considerable quantities of steroid hormone, but exerts little inhibitory effects on the transport of steroids into brain, whereas globulin-bound hormone does not appear to be transported into brain to a significant extent. Therefore, the hormone fraction in plasma that is available for transport into brain is not restricted to the free (dialyzable) fraction, but includes the larger albumin-bound moiety.

Resolution of the ATP-dependent proteolytic system from reticulocytes: a component that interacts with ATP.

Abstract: The ATP-dependent proteolytic cell-free system from reticulocytes has been resolved into three components, each of which is absolutely required for acid solubilization of 125I-labeled bovine serum albumin radioactivity. In addition to the previously reported heat-stable polypeptide [Ciechanover, A., Hod, Y. & Hershko, A. (1978) Biochem. Biophys. Res Commun. 81, 1100-1105], we now describe a protein of high molecular weight (approximately 450,000) that is labile at 42 degrees C. The extremely heat-labile factors is remarkably stabilized by ATP. GTP and CTP, which do not stimulate protolysis, do not stabilize this factor. Adenylate nucleotides such as ADP or the nonhydrolyzable beta,gamma imido or methylene analogues of ATP cause stabilization although they do not activate proteolysis. A third protein component of the protease system, stable at 42 degrees C, has been separated from the heat-labile species by salt precipitation. All three components are required with ATP for proteolytic activity, but thus far only the heat-labile factor has been shown to interact directly with ATP.

Carrier-Mediated Transport of Thyroid Hormones through the Rat Blood-Brain Barrier: Primary Role of Albumin-Bound Hormone*

Abstract: The transport of [l25I]T3., and [l25I]T4 through the brain capillary wall, i.e. the blood-brain barrier, was studied in barbiturate-anesthetized rats using a tissue-sampling-carotid injection technique. The percent extraction of unidirectional influx of thyroid hormone during a single pass through the brain was measured relative to a highly diffusible [3H]water reference. The Km of T3 transport was 1.1 μM; T3 transport was inhibited by T4 (Ki = 2.6 μM), rT3;, (Ki = 5.4 μM), and D-T3 but not by 1000 μM concentrations of tyrosine, leucine, or potassium iodide. Bovine albumin also inhibited blood-brain barrier transport of T3). The fractional inhibition of T3 transport by albumin was a measure of the binding of T3 by albumin in vivo, i.e. in the presence of a competing binding system, the BBB T3 carrier. The apparent dissociation constant (Kd) of albumin binding of T3 at the brain capillary level (76 μM) was 16-fold greater than the K3 of albumin binding of T3 in vitro (4.7 μM), as determined by equilibrium ...

Purification of Streptomyces chromofuscus Phospholipase D by Hydrophobic Affinity Chromatography on Palmitoyl Cellulose

Abstract: Phospholipase D [phosphatidylcholine cholinehydrolase, EC 3.1.4.4] excreted from Streptomyces chromofuscus was purified from the culture supernatant by precipitation with acetone and column chromatographies on palmitoylated gauze (Pal-G), DEAE-cellulose, and Sephadex G-150 with an overall recovery of 46% and 1000-fold increase in specific activity. The purified enzyme preparation showed a single band on sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. The enzyme had a molecular weight of about 50,000 by gel filtration on Sephadex G-150 or about 57,000 by SDS-polyacrylamide disc gel electrophoresis and an isoelectric point (pI) of pH 5.1 on isoelectric focusing. The enzyme hydrolyses lecithin, lysolecithin, sphingomyelin, and cephalin; the relative reaction velocities and Km's for choline-phospholipids were 87% and 1.43 mM for lecithin, 100% and 1.67 mM for lysolecithin, and 22% and 0.56 mM for sphingomyelin. The enzymatic reaction was optimal at pH 8, and its velocity was appreciably increased by either detergent (Triton X-100, deoxycholate), Ca2+ or both detergent and Ca2+. Diethyl ether stimulated the enzymatic activity by 30%; SDS and EDTA inhibited the activity. Bovine serum albumin, Triton X-100, and lipids (lecithin, lysolecithin, phosphatidic acid, lysophosphatidic acid, palmitic acid, and oleic acid) inhibited adsorption of the purified enzyme onto palmitoyl cellulose (Pal-C) and affected both the enzyme activity and stability: albumin and Triton X-100 increased the activity and enhanced the heat-stability; lysophospholipids decreased the activity but other lipids increased the activity; all the lipids lowered the heat-stability. The enzyme adsorbed on Pal-C was active, although its activity was about one-ninth of that of free enzyme, and was protected from heat-inactivation. Thus this enzyme appears to possess a hydrophobic site distinct from its catalytic site and to be adsorbed onto Pal-C through the hydrophobic site. Albumin, Triton X-100, and lipids seem to bind to the hydrophobic site and to have an appreciable effect on the enzyme activity and stability.

Direct demonstration of binding of a lysosomal enzyme, alpha-L-iduronidase, to receptors on cultured fibroblasts.

Abstract: Receptor-binding of "high-uptake" forms of lysosomal enzymes to human diploid skin fibroblasts had been predicted from the Michaelis--Menten kinetics of uptake of these enzymes [e.g., Sando, G.N. & Neufeld, E.F. (1977) Cell 12, 619--627]. We have now demonstrated such binding directly by using a sensitive assay for the bound enzyme. Cells deficient in alpha-L-iduronidase were detached from plastic dishes by mild trypsinization, allowed to recover, and used in suspension. They were incubated with urinary alpha-L-iduronidase at 0 degrees C for 90 minutes and then washed by centrifugation through concentrated bovine serum albumin; the activity of the cell-associated enzyme was measured with 4-methylumbelliferyl alpha-L-iduronide as substrate. A Scatchard analysis showed 14,000 binding sites per cell and a Kd of 1 x 10(-9) M for high-uptake alpha-L-iduronidase; binding of the low-uptake form was barely detectable. Mannose 6-phosphate, a known competitive inhibitor of uptake, inhibited the binding competitively, with Ki = 1 x 10(-4) M. Unexpectedly, mannose 6-phosphate greatly accelerated the dissociation of bound enzyme. During uptake of alpha-L-iduronidase at 35 degrees C, the receptors were regenerated every few minutes, even in the absence of protein synthesis.

Diethylstilbestrol and estradiol binding to serum albumin and pregnancy plasma of rat and human.

Abstract: The equilibrium binding of diethylstilbestrol (DES) and 17β-estradiol (E2) to plasma proteins has been characterized. DES exhibits a 10- to 20-fold greater binding affinity index for bovine serum albumin and rat plasma than E2. As expected, E2 gave high values for binding to plasma from pregnant mice or rats, reflecting the presence of α-fetoprotein. DES bound to these samples as it did to bovine serum albumin and rat plasma. These results suggested that DES interacts weakly with α-fetoprotein. This was verified by Scatchard plots of DES and E2 binding to rat and human pregnancy plasma. High affinity, low capacity binding was demonstrated with E2 but not with DES. The significantly lower binding of DES suggests that increased delivery of DES to the fetus may be at least partially responsible for the transplacental toxicity and carcinogenicity of DES.

Photo‐conjugation of 8–methoxypsoralen with proteins

Abstract: — 8–Methoxypsoralen (8–MOP) is shown to form a covalent conjugate with bovine serum albumin (BSA) by UVA irradiation in the presence of 02. The photoreaction is shown to involve oxidation of 8–MOP itself as a first step, producing an oxidation product which reacts readily with protein. Thus, this photoreaction appears to be completely different from the known 8–MOP photo-cycloaddition reaction with DNA. Among several proteins studied, human serum albumin, histone type 11, RNAse A and lysozyme also undergo photoinduced addition by 8–MOP. Chemical modifications of various amino acid residues in BSA revealed tyrosine-OH as one of the reaction sites. Irradiation (12 h) with UVA at 1.78 J/ds intensity resulted in approximately 1.5 mot of 8–MOP bound to 1 mol of BSA.

Measurement of 1,25-dihydroxycholecalciferol in man by radioimmunoassay

Abstract: 1 alpha, 25-Dihydroxycholecalciferol (1,25-(OH)2D3) has been measured in human serum by radioimmunoassay. The assay uses a high titre antiserum raised in sheep against 1,25-(OH)2D3-25-hemisuccinate, conjugated to bovine serum albumin. The sensitivity of the assay is 10 pg/tube. Other hydroxylated forms of vitamin D3 cross react with the antiserum and are therefore removed from serum extracts by chromatography on Sephadex LH 20 followed by high pressure liquid chromatography. The mean (+/- SEM) serum 1,25-(OH)2D3 concentration for a group of healthy adult subjects was 41 +/- 2.5 pg/ml. None was detected in anephric patients and the concentration was low or undetectable in patients with chronic renal failure.

Drug-carrier Property of Albumin Microspheres in Chemotherapy. II. Preparation and Tissue Distribution in Mice of Microsphere-entrapped 5-Fluorouracil

Abstract: Bovine serum albumin microspheres contatining 5-fluorouracil-6-3H were prepared by heating at 180° (or 150°, 100°) of 25% albumin solution in cottonseed oil emulsion. The shape of this microsphere was invariably spherical, and the average diameter was 0.66μ. After intravenous injection in mice, 5-fluorouracil-6-3H entrapped in albumin microspheres localized mainly in the liver, and the disappearance rate of radioactivity in microspheres from the tissue was very slow in comparison with that of free drugs. The microsphere might be delivered into reticuloendothelial system in the liver because ofits phagocytic activity, as well as the distribution following injection of albumin macroaggregates. Such preferential localization and sustained release of entrapped drugs suggested that albumin microspheres are useful as drug-carrier in chemotherapy.

Prolonged retention of estradiol by human breast cancer cells in tissue culture

Abstract: Conditions are described under which prolonged estradiol retention and estrogenic activity are observed in human breast cancer cells in tissue culture. The cells were incubated for three hr with a physiological concentration of [3H]estradiol (3 to 5 nM) and then were washed with 3 successive exchanges of medium 3, 17, and 24 or 48 hr following incubation with [3H]estradiol. The total wash period was 78 hr. The following parameters were monitored to assess the duration of estrogen action in MCF-7 human breast cancer cells in tissue culture; (a) the concentration of [3H]estradiol and [3H]estradiol metabolites in the media washes; (b) the intracellular concentration of [3H]estradiol and [3H]estradiol metabolites; and (c) the time course of estradiol-enhanced rates of radiolabeled thymidine incorporation. The [3H]estradiol concentration in the final medium wash was approximately 0.05 nM. The total intracellular concentration of tritium was about 50 nM prior to wash and 9 nM following 78 hr of wash. The intracellular concentration of specifically bound [3H]estradiol was initially 18 nM, and after 78 hr of wash, it was 2.8 nM. After 48 hr of wash, nearly all specifically bound [3H]estradiol was present in the nucleus. Following incubation of the cells with 5 nM estradiol and an identical wash procedure, estrogenic activity as measured by a stimulation of thymidine incorporation was observed throughout the 78 hr monitored. When 10(-6) M tamoxifen or 10(-7) M unlabeled estradiol was included in the medium washes, the washout of nonspecific binding was unaffected; however, specifically bound [3H]estradiol was essentially eliminated within 24 hr. When bovine serum albumin was included in the medium washes, total, nonspecific, and specific [3H]estradiol binding was reduced in a parallel and dose-dependent fashion. After 48 hr, cells washed with medium containing 3.5 or 7% bovine serum albumin contained one-tenth of the [3H]estradiol present in cells washed with medium alone. We conclude that medium exchanges alone do not effectively remove estradiol from MCF-7 cells, and suggest that estrogen retention by estrogen-responsive cells may mask in vitro assessments of such responsiveness in this and other systems. Inclusion of bovine serum albumin in the washes may alleviate this problem.

A simple, direct radioimmunoassay for plasma cortisol, featuring a 125I radioligand and a solid-phase separation technique.

Abstract: A simple, direct radioimmunoassay for cortisol in human serum and plasma is described. An antiserum, raised in sheep to a cortisol-3-(O-carboxymethyl)oxime/bovine serum albumin conjugate, is coupled to microcellulose. No extraction is required because plasma samples and standards are incubated with the antiserum and an 125I radioligand in a low-pH buffer, which denatures cortisol-binding globulins. The assay satisfies accepted validation criteria. In addition, results from the radioimmunoassay compare well with those obtained by a gas chromatographic-mass spectrometric technique (r = 0.968; FRIA = 0.97 FGCMS + 2.0 nmol/L). The latter procedure features the very high intrinsic specificity obtained by selected ion monitoring at high mass-spectrometric resolution (M/deltaM = 8500) with a Varian MAT-731 instrument. The simplicity of the radioimmunoassay procedure, with use of reagents prepared "in house," makes this a very practical and economical assay for use in the medium or large endocrine laboratory.

Transferrin can replace serum for in vitro growth of mitogen‐stimulated T lymphocytes

Abstract: Activation of human T cells by mitogens was compared in cultures containing serum, human serum albumin or purified human transferrin as growth support. The mitogenic effect of the lectins leucoagglutinin, concanavalin A and Wistaria floribunda agglutinin was measured as incorporation of [3H]thymidine into the cellular DNA of the lymphocytes. Three different preparations of transferrin were all able to fully substitute serum or serum albumin as growth promotors, when present at concentrations of 10 microgram/ml or more. A small contamination of transferrin in the human serum albumin preparations used was shown to be responsible for their growth-supporting effect, while no need for the presence of albumin itself could be demonstrated.

Large-scale production and partial purification of mouse immune interferon.

Abstract: Large-scale production of high-titered (102.2 to 104 U/ml) immune interferon (type II) was carried out in roller cultures of mouse spleen cells by using the T-cell mitogen staphylococcal enterotoxin A. Precipitation of 90% of this interferon by 55 to 80% saturated ammonium sulfate resulted in a 20-fold concentration and a two- to sixfold purification. After application of this interferon to either bovine serum albumin (BSA)-Affi-Gel 10 or hydroxylapatite columns, 100% of the interferon activity was recovered. By BSA-Affi-Gel 10 chromatography, 7% of the recovered activity was not bound, 45% was eluted with pH gradient 5 to 7, and 48% was eluted with 1 M NaCl. The pH- and salt-eluted interferons from the BSA-Affi-Gel 10 column were purified 62- and 390-fold, respectively, when compared with the starting materials. Rechromatography of the pH- and salt-eluted interferon peaks from the BSA-Affi-Gel 10 column did not alter their elution patterns. Stepwise elution of interferon from the BSA-Affi-Gel 10 columns with buffers of various pH and salt contents also resulted in greater than 300-fold purification. Specific activities of up to 2 × 105 U of interferon per mg of protein were attained with either elution procedure from BSA-Affi-Gel 10 columns. By hydroxylapatite chromatography, 5% of the recovered activity was not bound, 20% was eluted with a salt gradient, and 75% was eluted with 30% glycerin. Purification was 107- and 16-fold, respectively, for the two fractions. Ultrogel AcA 34 chromatography of the interferon resulted in two peaks of activity, a major one with a molecular weight of approximately 40,000 and a minor peak of molecular weight 70,000 to 90,000. Thus, by different types of chromatography, immune interferon was found to be heterogeneous.

Mitogenic effect of a growth factor derived from myelin on denervated regenerates of newt forelimbs

Abstract: Fibroblast growth factor injected into denervated early regenerates of adult newt forelimbs promotes mitotic activity in the dedifferentiated mesodermal cells. The effect is dose-dependent and is not produced with similar doses of cytochrome c, bovine serum albumin, or growth hormone. Epidermal growth factor has a mitogenic effect less than half as great as that of fibroblast growth factor. The results suggest the possibility of a role for myel in-derived growth factors in blastema formation.