José Uriel

Characterization of Natural Inhibitors of Trypsin and Chymotrypsin by Electrophoresis in Acrylamide-Agarose Gels

WE have detected natural inhibitors of enzymes m microgram quantities. In our method, after gel electrophoresis of a sample presumed to contain inhibitory activity, the slab of gel was incubated in a solution of the appropriate enzyme which then entered the gel by diffusion and formed a thin and homogeneous layer on its surface. After several minutes the gel was removed from the solution, allowed to stand until completion of the enzyme-inhibitor complex, and then transferred into a solution containing a chromogenic substrate for the enzyme used in the assay. In these conditions, the catalytic activity of the enzyme could be visualized, for the whole surface of the gel was stained, except for areas where the inhibitor was present.

Rat alpha-fetoprotein: isolation, characterization and estrogen-binding properties

Summary Rat α-fetoprotein (α-FP) was isolated from amniotic fluid by an immuno-chemical procedure using high capacity immunoadsorbents. The yield was about 75 p. cent of the initial content in α-FP. The isolated α-FP was found pure by electrophoretic and immunochemical criteria. A molecular weight of 72.000 daltons and a sedimentation coefficient of 4.5 S were estimated by SDS-acrylamide-agarose electrophoresis and sucrose gradient centrifugation, respectively. The latter procedure was also used to study the binding activity of α-FP toward several steroids. All the estrogens tested, estrone, estradiol, estriol and diethylstylbestrol, were bound. By equilibrium dialysis, the intrinsic association constant of pure α-FP was 1 × 108M−1 for estrone and 6 × 107M−1 for estradiol. One molecule of estrone and estradiol was bound per molecule of protein. No significant binding was observed with testosterone and progesterone. The specificity of the estrophilic activity of α-FP appears as a characteristic property of this protein. After electrophoresis of pure α-FP in acrylamide-agarose gels of low porosity (11 p. cent of acrylamide monomer), two closely migrating but distinct bands could be demonstrated. Both forms possess estrogen-binding activity and common antigenic properties. The same molecular heterogeneity of α-FP was observed in whole amniotic fluid.

Fatty acid metabolism in human lymphocytes. I. Time-course changes in fatty acid composition and membrane fluidity during blastic transformation of peripheral blood lymphocytes

The time-course changes in fatty acid composition of human T-lymphocytes during blastic transformation were analysed, as well as the variations in membrane fluidity determined by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), using a fluorescence-activated cell sorter. The more important changes observed, in activated relative to quiescent cells, started after 24 h and consisted in an increase in the proportion of oleic (18:1(n - 9)), docosapentaenoic (22:5(n - 3)) and docosahexaenoic (22:6(n - 3)) acids and a decrease in that of linoleic (18:2(n - 6)) and arachidonic (20:4(n - 6)) acids. This represented a relative increase of 26% for 18:1, 56% for 22:5 and 84% for 22:6 in peripheral blood mononuclear cells (PBMC) and 35%, 182% and 94%, respectively, in purified T-lymphocytes, both activated for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and 19%, respectively, for 72 h. The decrease in n - 6 fatty acids was of 42% for 18:2 and 14% for 20:4 in PBMC and 30% and phosphatidylethanolamine) rather than neutral lipids. The 18:1/18:0 ratio increased greatly in major cell phospholipids. The proportion of 20:4, 22:5 and 22:6 in phosphatidylinositol was not significantly altered after 72 h of activation. The molar ratio cholesterol/phospholipids was reduced in 72-h-activated lymphocytes (0.29) compared to quiescent cells (0.5). On the other hand, the stimulation of human T-lymphocytes caused a significant decrease in the order parameter (S) of DPH, according to the observed changes in lipid composition. After 72 h in culture, the S value for quiescent and stimulated T-lymphocytes was 0.530 and 0.326, respectively. In conclusion, the blastic transformation of human T-lymphocytes is associated with changes in lipid composition which modify the physical properties of their membranes. These modifications could modulate, in turn, the activity of membrane proteins implicated in the process of blastic transformation.

Affinity chromatography of human, rat and mouse α-fetoprotein on estradiol-sepharose adsorbents

Rat and mouSe serum o-fetoprotein (oFP) possesses high binding affinity for estrogens as demonstrated by biochemical [l-4] and immunological [ 51 methods. The results concerning human serum aFP are controversial. By either equilibrium or sucrose gradient centrifugation no significant estrogen-binding capacity could be demonstrated [3,4]. Gel diffusion immunoprecipitates of serum (rFP from a patient bearing primary liver cancer, showed, however, estrogenbinding activity [5] and, more recently, the specific binding of human serum aFP to estradiol-Sepharose columns was reported, although attempts to eluate the olFP from the adsorbent were unsuccessful [6]. In the present paper we describe a single procedure to isolate rat and mouse aFP from amniotic fluids after adsorption on estradiol-Sepharose beads and elution with saturated solutions of estrone in 15% aqueous dioxane. We also report preliminary results on the chromatographic behaviour of rat and mouse, as well as human (wFP upon insolubilized estradiol adsorbents. The results obtained provide further evidence that all three oFP possess estrogen-binding properties, although the proportion of active versus inactive aFP molecules present in a biological fluid varies greatly from one species to another.

Fatty acids bound to α-fetoprotein and albumin during rat development

Abstract The time-course levels and composition of the fatty acids bound to rat α-fetoprotein (AFP) and albumin from several sources, were determined throughout development, and related to the intake of lipids from milk and the compositional changes in brain and liver fatty acids. The major fatty acids bound to AFP were polyunsaturated and mainly docosahexaenoic acid (22:6(n − 3)), either from fetal serum (23.1%) or whole fetuses (21.6%), whereas palmitic (34.1%) and oleic (29.9%) acids were the main acids bound to albumin from the same sources. Amniotic fluid AFP contained less fatty acids (0.8 mol/mol protein) than that of fetal serum (1.4 mol/mol protein), and especially noticeable was a reduced amount of 22:6 (9.6%). Both AFP-concanavalin A microforms showed identical fatty acid composition. Levels of 22:6 bound to AFP decreased quickly after birth until a minimum at 8–10 days, increasing moderately thereafter. This minimum is coincident in time with a maximal accumulation of this fatty acid by brain and a loss of 22:6 by liver. Except for colostrum, levels of 22:6 in milk lipids were low and fairly constant, but always greater than those of its precursor, linolenic acid (18:3 (n − 3)). These results support a specialized role of AFP in the plasma transport and tissue delivery of polyunsaturated fatty acids, and mainly docosahexaenoic acid.

Two high affinity estrogen binding proteins of different specificity in the immature rat uterus cytosol

Abstract The presence of two high affinity estrogen binding proteins in the uterine cytosol of the immature rat has been observed. Besides the 8 S cytosol estrogen receptor , there is a 4–5 S fraction binding estradiol and estrone with a large capacity. In fact, the two binding systems have a different affinity for estradiol and estrone, the receptor binding more the former and the 4–5 S fraction more the latter. Exposure of the cytosol to specific anti-α1-Fetoprotein antibodies suppresses a large part of the 4–5 S binder, if not the totality. Moreover the estrogen binding 4–5 S fraction decreases with increasing age until puberty, while the receptor increases. These results suggest therefore that the estradiol-estrone binding 4–5 S peak of the uterine cytosol is mainly made up of Estradiol Binding Plasma Protein-α1-Fetoprotein (EBP-AFP). Also they confirm that “cytosol” should be taken as an operational fraction which may include extracellular components. During the course of these experiments, it has been observed that the increase of the estradiol receptor is more rapid than the other uterine cytosol proteins until the 8th day, and that there is a second period of growth when it follows the development of the uterus and of the animal, as if it had reached a constant number of binding sites per cell.

Fatty acid metabolism in human lymphocytes. II. Activation of fatty acid desaturase-elongase systems during blastic transformation

The fatty acid desaturation-elongation ability of human T-lymphocytes during blastic transformation was determined both by gas-liquid chromatography and incubation with radiolabeled precursors. Human peripheral blood mononuclear cells (PBMC) were activated with phytohemagglutinin (PHA) and cultured in media supplemented with different fatty acids (18:0, 18:1(n - 9), 18:2(n - 6), 18:3(n - 3) and 20:4(n - 6)) at a final concentration of 30 microM. All the fatty acids added were elongated by activated PBMC and the maximal activity was observed on 20:4(n - 6) (a 25% of conversion to 22:4(n - 6)). Supplementation with stearic acid increased the proportion of oleic (from 21.4% to 23.7%) and eicosaenoic (from 3.1% to 5.7%) acids in cellular lipids, indicating the existence of a delta 9-desaturase activity. Supplementation with linoleic and linoleic acids increased slightly the cell content in their more unsaturated derivatives. Direct measurement of desaturase activities was performed by incubating quiescent and activated PBMC with [1-14C]stearic, [1-14C]linoleic and [1-14C]linolenic acids. Quiescent cells exhibited a very low delta 9-desaturase and no sign of delta 6-desaturase activity. A moderate and progressive activation of delta 9-, delta 6- and delta 5-desaturases was observed during blastic transformation of human PBMC. Up to 8% of 18:0 was converted to monoenes, 4% and 1.5% of 18:2(n - 6) was converted to trienes and tetraenes, respectively, and 14.5% of 18:3(n - 3) was converted to pentaenes. The maximal relative activities were found after 48 h of PHA-stimulation for delta 9-desaturase (around 90 pmol of 18:0 converted per 10(6) cells in the last 24 h) and at 72 h for delta 6- and delta 5-desaturases (around 75 and 140 pmol of 18:2 and 18:3, respectively, converted per 10(7) cells in the last 24 h). Although these activities are not enough to explain all the changes in fatty acid composition of human PBMC during blastic transformation, they may contribute to a more controlled cell phospholipid composition.

Trypsin inhibitor from cow colostrum. Isolation, electrophoretic characterization and immunologic properties.

Trypsin inhibitor from cow colostrum has been purified by affinity chromatography of colostral proteins on insolubilized trypsin. The method described compares favourably, in both simplicity and yield, with previous methods developed for the isolation of this inhibitor. Gel electrophoresis followed by characterization of antitrypsin activity allows the demonstration of four molecular forms of bovine colostral trypsin inhibitor in both crude colostral whey and purified preparations of the inhibitor. Immunoelectrophoresis of each of these materials with antisera specific for this inhibitor reveals a single precipitation line of broad anodic mobility. By immunodiffusion tests, the precipitation lines in preparations of purified inhibitor and colostral whey appear immunologically identical. In contrast, absence of crossed reactivity was observed between bovine colostral trypsin inhibitor and trypsin inhibitors of bovine serum. This strongly suggests the high specificity of this inhibitor as a colostral and milk constituent.

Alpha-Fetoprotein and Albumin Uptake by Mouse Tissues during Development

We have studied the evolution of the incorporation of alpha-fetoprotein (AFP) and albumin by mouse tissues from fetal to adult life. Mice were injected with 125I-labelled AFP or albumin and, 3 h later, blood and organs were removed and analyzed for their radioactive protein content. Results showed that all immature tissues examined took up both AFP and albumin from blood. AFP uptake was higher during fetal and early postnatal life and decreased with age. In general, the relative uptake of albumin was lower than that of AFP, and the time-course incorporation of both proteins was parallel in brain, liver and kidney. In other tissues such as white adipose tissue, brown adipose tissue and skin. AFP uptake decreased while albumin uptake remained almost constant or increased with age. In the fetal period, the strong AFP uptake in white adipose tissue contrasted with the much lower albumin incorporation by this tissue. Autoradiographs from sections of organs and entire animals confirmed the cytoplasmic localization of AFP and albumin. We conclude that AFP and albumin found in developing tissues, except for fetal liver and yolk sac, proceed mostly from blood uptake. These results agree with recent experimental data suggesting that the major physiological role of AFP is the transport and delivery of polyunsaturated fatty acids to developing tissues.

Carrier-protein-mediated enhancement of fatty-acid binding and internalization in human T-lymphocytes

Albumin and alpha-fetoprotein (AFP) are members of a multigene family which also includes vitamin-D-binding protein. Previous work in our laboratory has provided experimental support for the suggestion that the entry of unsaturated fatty acids into growing, normal and neoplastic cells may be regulated by AFP. In the actual study we have examined the role of human serum albumin (HSA) as a carrier protein, when compared to AFP, on the uptake (binding and internalization) of fatty acids by resting and PHA-activated human lymphocytes. Radioiodinated human HSA and tritiated oleic and arachidonic acids were used under different experimental conditions to follow the binding of the protein and fatty acids (FA) to cells. Time-course uptake at 4 degrees C of HSA and of oleic and arachidonic acids bound to HSA (FA/HSA molar ratio = 1) by either resting or activated T-lymphocytes exhibited a steady state of binding. The amount of FA bound was much greater than the corresponding amount of HSA, suggesting that T-lymphocytes bear distinct binding sites for albumin and fatty acids. A saturable process of FA binding was observed at constant unbound FA concentration in the incubation medium when the HSA-to-FA molar ratio was fixed at 1 and the concentrations of both HSA and FA were increased simultaneously. This saturable component of binding reflects an intrinsic regulatory effect of HSA, probably operating throughout the interaction of the protein with specific cell receptors. At varying unbound FA concentrations, binding curves showed two distinct components: a non-linear portion which could indicate the presence of a saturable process operating at low concentrations of unbound, free FA, followed by a second part which increased linearly with the concentration of unbound FA. The amount of FA bound at 4 degrees C and bound and internalized at 37 degrees C by both types of cell was considerably higher in the presence than in the absence of carrier proteins. On the contrary, carrier proteins were without effect on the distribution pattern of internalized oleic or arachidonic acid. Taken together, these observations suggest that: (i) the binding and entry of FA into cells is enhanced by the two carrier-proteins at low concentrations of free, unbound fatty acids in the vicinity of the cell surface, and (ii) fatty-acid uptake seems regulated by a dual-receptor mechanism involving HSA and/or AFP receptors as well as plasma-membrane FA-binding proteins.