The modified method for isolation and culture of highly homogeneous Leydig cell population from rat testes

A modified method for isolation and culture of a pure population of rat Leydig cells is described. For obtaining crude interstitial cell suspension, decapsulated testes were dispersed in 0.02% collagenase solution in Ca2+, Mg2+--free Hanks medium for 1 hour. Then, approx. 5 X 10(7) cells were centrifuged in 10-90% discontinuous, isoosmotic Percoll gradient at 3000 g for 20 min. The cells from eight fractions obtained were collected and cultured in Eagle's MEM for 4 days. Using morphological methods, 1.059-1.070 g/ml density fraction contained 97% and 1.070-1.080 g/ml fraction contained 90% viable Leydig cells. The cells secreted testosterone to the culture medium and responded to LH stimulation with over four-fold increase in hormone secretion.     

Guinea pig adrenocortical cells: in vitro characterization of separated zonal cell types

This paper reports a quick, relatively simple and reproducible technique for obtaining populations of zona fasciculata and zona glomerulosa cells up to 80-90% pure, which can be maintained in vitro for study of adrenocortical cell function. Isolated guinea pig adrenocortical cells were separated on a 1-28% bovine serum albumin/Ca++, Mg++-free buffer gradient (wt/vol at 4% increments) using equilibrium density centrifugation (570 g, 30 min). Over 60% of the 8 x 10(5) viable cells/adrenal obtained in the total isolate were recovered after separation. 80% of the zona glomerulosa cells were found in the lower three bands of the gradient. 78% of the zona fasciculata cells were found in the top three bands. Of the cells in the first two bands, 78-91% were zona fasciculata cells, whereas of the cells in the bottom two bands 92-95% were zona glomerulosa cells. The cells retained the morphological characteristics of cells in situ and could be maintained in vitro for periods up to 11 d. They produced a wide variety of steroids, cortisol, corticosterone, aldosterone, 11-beta- hydroxyandrostenedione, deoxycortisol, deoxycorticosterone, cortisone, 18-hydroxycorticosterone, and a product tentatively identified as dehydroepiandrosterone, and they responded to ACTH in a dose-responsive manner with enhanced levels of steroid output. Zona glomerulosa- enriched populations differed from zona fasciculata-enriched populations in their abundant production of aldosterone and in the pattern of steroid production. None of the cultures responded to angiotensin II (100 pg/ml) with increased steroid production.     

Receptor-mediated endocytosis of polymeric IgA and galactosylated serum albumin in rat liver. Evidence for intracellular ligand sorting and identification of distinct endosomal compartments

Rat polymeric IgA (pIgA) and galactosylated bovine serum albumin (GalBSA), once injected to rats, are avidly taken up by hepatocytes via receptor-mediated endocytosis. Of injected pIgA, 64% was transferred undigested into bile within 3 h, with a peak at 30-45 min. GalBSA was essentially digested in lysosomes. By electron microscopy using ligand-peroxidase conjugates, both ligands were internalized through coated pits/coated vesicles into similar electron-lucent vesicles and tubules. Subsequently, pIgA remained mostly associated with small vesicles clustering around and fusing with bile canaliculi, while GalBSA was predominantly found in large, heterogeneous endocytic structures and in lysosomes. By subcellular fractionation, they were associated at 3 min after injection with structures that similarly sedimented in the P fraction (250 000 - 3 X 10(6) X g X min) and equilibrated at densities of about 1.13 g/ml in sucrose gradients. At 10 min and 20 min, pIgA distribution remained mostly in the P fraction at the same equilibrium density. A minor component of the pIgA distribution was found at the density of lysosomes, but contrary to lysosomal enzymes, its distribution was not affected by Triton WR 1339. In contrast to pIgA, GalBSA was progressively recovered in the L fraction (33 000 - 250 000 X g X min) with organelles equilibrating around 1.11 g/ml, and, by 20-45 min, was found in the ML fraction (10 000 - 250 000 X g X min), around 1.20 g/ml, i.e. in lysosomes. Chloroquine did not reduce the efficiency but delayed the secretion of pIgA into bile. Similarly, it did not affect the uptake of GalBSA but apparently delayed GalBSA transfer along successive populations of host organelles. The low density, GalBSA-containing structures were devoid of proteolytic activity. Anti-secretory components IgG and F(ab')2 were selectively excreted into bile, partially or totally as compounds of lower molecular mass. These antibody fragments probably result from a disulfide reduction activity along the pIgA pathway. In conclusion, our data (a) strongly suggest that pIgA and GalBSA are sorted between 3 min and 10 min after injection in non-lysosomal acidic organelles, (b) identify two successive and physically distinct endosomal populations containing GalBSA, and (c) provide the first evidence for a disulfide reduction activity along the transcytotic pathway of rat hepatocytes.     

Interstitial cell heterogeneity in rat testes. II. Purification of cells by Percoll and metrizamide gradient centrifugation with preferential localization of gonadotropin binding sites in light cell fraction and hormone-induced steroidogenesis in heavier cell fraction

The ability of 125I-labeled human chorionic gonadotropin (125I-labeled hCG) to bind and stimulate steroidogenesis was studied in light cells (density, 1.053-1.065 g/cm3) and heavier cells (density, 1.090-1.110 g/cm3) purified from collagenase-dispersed rat testicular interstitial cells by unit gravity sedimentation (Bhalla, V.K., Rajan, V.P., Burgett, A.C., and Sohal, G.S. (1987) J. Biol. Chem. 262, 5313-5321). Preferential localization of gonadotropin binding sites was demonstrated on light cells, and the heavier cells produced testosterone in response to hCG without occupancy of high affinity (Kd = 2.02 X 10(-10) M) binding sites. In this study, established methods for interstitial cell purification involving gradient centrifugation were utilized to demonstrate the cell heterogeneity. Light cells bound hCG with high affinity (Kd = 3 X 10(-10) M) without manifestation of steroidogenic response. The heavier cells responded to hCG with elicitation of steroidogenesis, but the occupancy was negligible. Stimulation of steroidogenesis by hCG in heavier cells was dose and time dependent. Dibutyryl and bromo cyclic AMP (1 mM) also promoted steroidogenesis comparable to a level stimulated by the tropic hormone (700% stimulation). The concept of spare receptors was tested in purified cell fractions. Upon cell purification, no saturable high affinity binding sites were observed in the heavier cell fraction. Autoradiographic analyses at the electron microscopical level supported this conclusion. Our data suggest that target cell activation is not preceded by hormone occupancy of high affinity binding sites. A model for defining the functional domains of the physiological receptor for hCG is presented.     

Bovine adrenocortical cells exhibit high affinity transforming growth factor-beta receptors which are regulated by adrenocorticotropin

Transforming growth factor beta (TGF-beta) at picomolar concentrations has been previously shown to induce striking alterations of bovine adrenocortical cell differentiated functions, without detectable effect on growth activity (Feige, J.J., Cochet, C., and Chambaz, E. M. (1986) Biochem. Biophys. Res. Commun. 139, 693-700; Hotta, M., and Baird, A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7795-7799). Adrenocortical cells in culture could bind 125I-labeled TGF-beta through at least two different binding systems. The highest affinity TGF-beta binding exhibited a Kd value of 5.7 X 10(-10) M and a calculated capacity of about 100,000 sites/cell, while the low affinity system yielded values of 4.3 X 10(-8) M and 2 X 10(6) sites/cell, respectively. The 125I-labeled TGF-beta bound to adrenocortical cells could be cross-linked using disuccinimidyl suberate and subsequent electrophoretic analysis revealed that TGF-beta was associated with two major cell components of about 280 kDa and 70-75 kDa, respectively, the latter one being resolved as a labeled doublet. Thus bovine adrenocortical cells exhibit a TGF-beta receptor similar to that defined by Massagué and co-workers (Cheifetz, S., Like, B., and Massagué, J. (1986) J. Biol. Chem. 261,9972-9978) in other cell types. Various growth factors, including fibroblast growth factor, as well as established hormonal activators of adrenocortical cell differentiated functions, such as angiotensin II and adrenocorticotropin, were examined as to their effect on TGF-beta receptor activity. A striking increase in the number of high affinity TGF-beta receptors was selectively elicited by ACTH in the nanomolar concentration range. This effect was time- and dose-dependent and was mimicked by cell treatment with dibutyryl cyclic AMP or forskolin. However, the ACTH-induced increase in receptor number was not impaired when protein synthesis was blocked. It is concluded that bovine adrenocortical cells are typical target cells for TGF-beta. This endocrine system represents a model in which, for the first time, the level of TGF-beta receptor is shown to be under hormonal regulation through a cyclic AMP-dependent pathway.     

Cellular effects of endotoxin in vitro: mobility of endotoxin in the plasma membrane of hepatocytes and neuroblastoma cells

Lipopolysaccharide labeled with fluorescein isothiocyanate (FITC-LPS) was used to examine interactions between endotoxin and plasma membrane in isolated rat hepatocytes and mouse neuroblastoma NB41A3 cells. At the same endotoxin to cell ratio, hepatocytes bound more toxin than did neuroblastoma cells. At a dose of 12 micrograms/mg dry wt, a bound mobile fraction of between 60 and 75% of FITC-LPS was found on hepatocytes at 25 degrees C with a lateral diffusion coefficient (D) of 4.0 X 10(-9) cm2/s. In neuroblastoma cells, the mobile fraction was larger (85-90%), with D 1.0 X 10(-8) cm2/s. D was temperature-dependent between 10 and 37 degrees C and increased from 1.8 X 10(-9) to 1.0 X 10(-8) cm2/s in hepatocytes and from 9.4 X 10(-9) to 1.9 X 10(-8) cm2/s in neuroblastoma cells. In both types of cell, nonviable (cells which did not exclude Trypan blue) as compared to viable cells showed different recovery patterns and 100% of the probe molecules were mobile. These results suggest that: (1) endotoxin binding to mammalian cells consists of two subpopulations with different mobilities; (2) binding of the immobile fraction is dependent on cellular integrity; and (3) the differences in binding, lateral mobility, and size of the immobile fraction in hepatocytes and neuroblastoma cells may be due to variations in membrane composition and/or number of binding sites.     

Cytotoxic and suppressor activity of human peripheral blood lymphocytes stimulated by interleukin-2 and phytohemagglutinin before and after fractionation in the percoll gradient

The cultivation of peripheral blood lymphocytes (PBL) obtained from patients with colorectal or bladder carcinoma and melanoma and from healthy donors in the presence of interleukin-2 (IL-2) and PHA resulted in the induction of cytotoxic activity against autologous and/or allogeneic tumour cells in 12 out of 13 patients and in 10 out of 10 donors. A higher level of cytolytic activity was achieved when PBL were separated by means of Percoll density gradient (1.077; 1.067 and 1.056 g/ml) centrifugation and the cells of fraction II (1.077-1.067 g/ml) were employed in the experiment, the level of cytotoxicity being elevated in all cases (1.7-fold elevation in donors and 2-fold elevation in patients on the average). The addition of fraction I (1.067-1.056 g/ml) to fraction II prevented (PHA + IL-2)-mediated induction of cytotoxic activity in all the patients, but in 4 out of 10 donors, i.e. cells of fraction I expressed a suppressor activity. The immunofluorescent analysis has shown that fraction II was enriched by T cells (92%) and depleted of monocytes (7%), as compared to unseparated PBL (66% and 27%, respectively). On the contrary, fraction I was characterized by a decreased T cell ratio (36%) and an increased monocyte level (up to 69%).     

Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.

The purpose of this experiment was to characterize the high density lipoproteins (HDL) as a function of hydrated density. HDL was subfractionated on the basis of hydrated density by CsCl density gradient centrifugation of whole serum or the d 1.063-1.25 g/ml HDL fraction isolated from three men and three women. Apolipoprotein A-I and A-II quantitation by radial immunodiffusion showed that the A-I/A-II ratio varied with the lipoprotein hydrated density. The A-I/A-II molar ratio of HDL lipoproteins banding between d 1.106 and 1.150 g/ml was nearly constant at 2.2 +/- 0.2. In the density range 1.151-1.25 g/ml the A-I/A-II ratio increased as the density increased. On the other hand, in the density range between 1.077 and 1.105 the A-I/A-II ratio increased as the density decreased, ranging from 2.8 +/- 0.5 for the d 1.093-1.105 g/ml fraction to 5.6 +/- 1.3 for the d 1.077-1.082 g/ml fraction. The d 1.063-1.076 g/ml fraction and the d 1.077-1.082 g/ml fractions had comparable A-I/A-II ratios. Serum and the d 1.063-1.25 g/ml HDL fraction exhibited similar trends. The cholesterol/(A-I + A-II) ratio decreased as the density increased in all 12 samples (six serum and six HDL) examined. Gradient gel electrophoresis of the density gradient fractions showed that as the density increased from 1.063 to 1.200 g/ml the apparent molecular weight decreased from 3.9 x 10(5) to 1.1 x 10(5). HDL subfractions with the same hydrated densities had comparable molecular weights and A-I/A-II and cholesterol/(A-I + A-II) ratios when isolated from men or women. HDL contains subpopulations that differ in the A-I/A-II molar ratio.-Cheung, M. C., and J. J. Albers. Distribution of cholesterol and apolipoprotein A-I and A-II in human high density lipoprotein subfractions separated by CsCl equilibrium gradient centrifugation: evidence for HDL subpopulations with differing A-I/A-II molar ratios.     

Transforming growth factor beta treatment decreases ACTH receptors on ovine adrenocortical cells

Transforming growth factor beta (TGF beta) is a potent inhibitor of adrenocortical cell differentiated functions, whereas corticotropin (ACTH) is the main physiological hormone which acts positively on these functions. We have studied the effects of both TGF beta and ACTH on ovine adrenocortical cell ACTH receptors. Ovine adrenocortical cells contained specific high affinity (Kd = 2.7 +/- 1.6 x 10(-10) M) and low capacity (1190 +/- 120 sites/cell) ACTH receptors. Pretreatment of cells with TGF beta resulted in a time- and dose-dependent (ED50 = 50 pg/ml) decrease of 125I-ACTH1-39 binding. The observed decrease in ACTH binding was due to a 2-3-fold decrease in the number of binding sites without modification of the binding affinity. On the contrary, pretreatment of cells with ACTH caused a 4-4.5-fold increase in the number of ACTH binding sites without an effect on the Kd. When cells were pretreated with both ACTH and TGF beta, TGF beta blocked completely the positive trophic effect of ACTH on its own receptors. The variations in ACTH receptor number were associated with parallel changes on acute ACTH-induced cyclic AMP production. Thus, the effects of TGF beta on ACTH receptor content are likely another important negative action of this peptide on adrenocortical cell differentiation. Moreover, these results suggest that regulation of ACTH receptor number may be one mechanism by which hormones and growth factors control adrenocortical differentiation.     

Correlation between trypsin binding to a specific receptor and prostacyclin production in cultured vascular endothelial cells

The correlation between the binding and processing of trypsin and its effect on prostacyclin (PGI2) production in cultured adult bovine aortic endothelial (ABAE) cells was studied. ABAE cells demonstrated an ability to produce PGI2 in a dose-response manner to trypsin at the range of 0.1-2.0 micrograms/ml with a saturation at a concentration of 1 microgram/ml. Likewise, 125I-trypsin binding to the cultured cells increased in a dose-response way and reached saturation at a concentration of about 1 microgram/ml; 125I-trypsin was bound to a specific high-affinity cell-surface receptor with a dissociation constant (Kd) of 1.5 X 10(-8) M and each of the confluent ABAE cells has about 1.2 X 10(5) such receptors sites. The cell-surface receptor for trypsin displayed specific characteristics and an excess amount of unlabeled trypsin successfully abolished 125I-trypsin binding while thrombin in excess failed to compete for 125I-trypsin binding. Only a small fraction of the cell-surface-bound 125I-trypsin was internalized and subsequently degraded by ABAE cells as compared to the process of 125I-trypsin internalization by human skin fibroblasts (HSF). This study demonstrated that the stimulatory effect of trypsin on prostacyclin production and release by ABAE cells might be mediated by a specific cell-surface receptor for trypsin on these cells distinct from the thrombin receptor.     

Effects of mitomycin C on sister chromatid exchange in normal and Bloom's syndrome cells

Bloom's syndrome lymphocytes, which are characterized by a high incidence of sister chromatid exchanges (SCE: 80.6 per cell), were treated with mitomycin C (MMC) and the effect of the chemical on SCE frequency compared with that in normal cells. Raising the concentration of MMC from 1 X 10(-9) to 1 X 10(-7) g/ml led to about 10-fold increase (61.7 SCE per cell) in the SCE frequency over the base line in normal lymphocytes (6.4 SCE per cell), though chromosome aberrations remained at a relatively low frequency. MMC caused about a two-fold rise in SCE in cells of Bloom's syndrome (128.8 SCE at 10(-9) g/ml; 139.3 SCE at 10(-8) g/ml). The frequency of chromosome aberrations in Bloom's syndrome cells at concentrations of MMC of 1 X 10(-9) and 1 X 10(-8) g/ml was 0.350 and 0.825 per cell, respectively, and low when compared to the increased number of SCE. The increased frequency of SCE in normal and Bloom's syndrome cells is in contrast to the reported findings with cells from Fanconi's anemia and xeroderma pigmentosum. The distribution of SCE in MMC-treated normal cell correlates with that of spontaneous SCE in cells of Bloom's syndrome.     

The Euglena gracilis chloroplast genome: analysis by restriction enzymes.

1. Chloroplast DNA was isolated from autotrophically and mixotrophically grown Euglena gracilis cells. 2. Aliquots of chloroplast DNA were mechanically degraded to an average molecular weight of 4-7 X 10(6) and G+C-rich DNA fragments (density 1.701 g/cm3) were separated from the bulk DNA (density 1.685 g/cm3) using preparative CsCl density gradients. 3. Total chloroplast DNA and its DNA subfractions, which first were characterized with respect to average G+C content and hybridization capacity for chloroplast rRNA, were hydrolysed with restriction endonucleases (endo R-EcoRI, end R-HindII, endoR-HindIII, endo R-HindII+III, endoR-Hpal, endo R-HpaII and endoR-HaeIII). The fragments were separated on gels under a variety of electrophoretic conditions. 4. With each enzyme tested, a rather large number of bands was obtained. In all cases, different banding patterns were obtained for total DNA, and the DNA subfractions. 5. Chloroplast DNA from autotrophically and mixotrophically grown cells gave identical banding patterns. 6. Digestion of total DNA with the endoR-HaeIII yielded 51-52 fragments separated in the gels in a total of 36 bands of which 11-12 bands were composed of 2-3 fragments as estimated by densitometry. The molecular weights of all fragments combined was 87 X 10(6) or 95% of the genome (92 X 10(6)). 7. Chloroplast RNA hybridized to 5.1% with total chloroplast DNA, equal to three RNA cistrons per genome (Mr92 X 10(6)). These cistrons are located on seven different types of endo R-HaeIII fragments. The hybridising fragments are preferentially found in the G+C-rich subfraction and in bands which are composed of 2-3 fragments.     

Total and free testosterone in plasma of hypo- and agonadal men.

Plasma total testosterone (T), apparently free T and testosterone binding globulin (TeBG) capacity determined in 14 normal men aged 30-40 years were 461 +/- 100 ng/100 ml, 9.4 +/- 3.0 ng/100 ml and 5.7 +/- 1.9 X 10(-8) M, respectively, whereas in 16 hypogonadal men the corresponding values were 38.6 +/- 27.2 ng/100 ml, 0.47 +/- 0.41 ng/100 ml and 10.4 +/- 3.4 X 10(-8) M showing the TeBG capacity significantly higher (p less than 0.001) in hypogonadal than in normal men. Treatment of 5 hypogonadal subjects with 250 mg testosterone enanthate plus 50 mg testosterone propionate decreased (p less than 0.001) the TeBG level from 14.7 +/- 2.5 X 10(-8YM to 8.3 +/- 1.4 X 10(-8) M on day 8 after a single injection. According to this difference in TeBG, the free T fraction in plasma rose from 0.94% to 1.9% of the total T concentration. These results suggest that alteration of total plasma T affected the TeBG capacity. Decreased T levels raised and increased T concentrations suppressed TeBG, but with a delayed response to the changed T concentrations. The initial mean values in 12 patients with prostatic cancer aged 60-74 years were 397 +/- 165 ng/100 ml, 4.05 +/- 1.8 ng/100 ml and 11.9 +/- 3.3 X 10(-8) M, respectively. The TeBG capacity in these patients was significantly higher and the free T concentration significantly lower (p less than 0.001) than those of the younger normal males. After treatment with 12 g diethylstilbestrol diphosphate and orchidectomy, the TeBG increased to 33.3 +/- 13.1 X 10(-8) M and the plasma free T concentration decreased to the minimal value of 0.053 +/- 0.04 ng/100 ml.     

Characterization of high density lipoprotein binding activity in rat adrenocortical cells

Rat adrenocortical cells take up high density lipoprotein cholesterol for use as steroidogenic substrate. To better understand this unique uptake process, we have first characterized HDL binding. Infusion of human 125I-labeled HDL into rats pretreated with 4-APP demonstrated that the adrenal and ovary accumulate HDL in a saturable fashion in vivo. Subsequent studies using isolated rat adrenocortical cells demonstrated that cellular uptake of HDL is comprised of two events. One event is characterized by reversible membrane binding and is complete by 60 min (t1/2 = 20 min). The second event is marked by irreversible apoprotein accumulation which continues for at least 3 hr. Reversibly bound material exhibits the same apoprotein distribution as unincubated HDL. Irreversible accumulation could not be attributed to internalization or lysosomal accumulation inasmuch as it also occurred with partially purified plasma membranes and was not enhanced by addition of chloroquine. Reversible binding of human HDL3 exhibited a saturable dependence on concentration (Kd = 27 micrograms protein/ml; N = 3.0 X 10(6) sites/cell) similar to that previously reported for rat liver, ovary, and testis. Cell accumulation of HDL decreased by over 80% at 4 degrees C compared to 37 degrees C, did not require calcium, and was not diminished by prior cell treatment with trypsin or pronase. These results indicate that rat adrenocortical cells possess plasma membrane recognition sites for HDL with different properties than those of the LDL receptor. Moreover, adrenal accumulation of HDL apoproteins does not lead to secondary lysosome formation.     

Biochemical and immunological characterization of estrogen binding components in human neoplastic adrenocortical tissues

The estrogen binding components in human adrenocortical tissues were examined. Two adrenocortical cancer cytosols were found to contain the binder with a relative low affinity (Kd 5 X 10(-9) M) for estradiol. The association of [3H]estradiol to these cytosols was inhibited by a large dose of unlabeled estrone, estradiol or estriol, but neither by diethylstilbestrol nor by dihydrotestosterone. Incubation of cultured cells derived from these cancers with [3H]estradiol also showed the presence of this low-affinity estradiol binder. The addition of bovine serum albumin into these cytosols surprisingly resulted in a marked increase in estradiol binding capacity in a concentration-dependent manner. This component sedimented at 5 S in the low salt sucrose density gradient. This binding ability was found to be heat-labile in the absence of estradiol, but preformation of complexes with estradiol markedly stabilized its binding ability against thermal inactivation. In addition, experiments using monoclonal antibodies to human estrogen receptor revealed that the estrogen binder from one adrenocortical cancer cytosol shared antigenic determinants with human estrogen receptor. These results suggest that the unique estrogen binder in some adrenocortical cancer has the characteristics similar to estrogen receptors in terms of thermal stability and immunological cross-reactivity to antibodies.     

Characterization of equine plasma lipoproteins after separation by density gradient

1. Plasma lipoproteins from six thoroughbred horses were separated by density gradient ultracentrifugation. For each sample, lipoprotein bands were visualized by means of a prestained plasma control and characterized by electrophoretic, chemical and morphological analysis. 2. Very low density lipoproteins (VLDL) were isolated at d less than 1.018 g/ml. 3. Two clearly resolved bands were detected in the low density lipoprotein fraction (LDL). The density limits were evaluated as follows: LDL1(1.028 less than d less than 1.045 g/ml) and LDL2(1.045 less than d less than 1.070 g/ml). Marked differences were observed in the chemical composition and particle size of LDL1 and LDL2 fractions. 4. High density lipoprotein fraction (HDL) was usually isolated as a single band, distributed over the range 1.075 less than d less than 1.180 g/ml. However, chemical composition and particle size revealed heterogeneity in HDL subfractions. 5. The density limit of LDL and HDL bands varied in each animal, indicating differences in equine lipoprotein distribution.     

Protein interaction with immobilized ligands: quantitative analyses of equilibrium partition data and comparison with analytical chromatographic approaches using immobilized metal affinity adsorbents

Quantitative or analytical affinity chromatography has been successful primarily for the analysis of biologically determined macromolecular affinity relationships. Quantitative approaches are also needed to better characterize simpler, chemically defined immobilized ligands with potential for selective interaction with specific, predetermined protein surface groups. Protein interaction with immobilized metal is a rather selective and versatile, high-affinity adsorption technique for which there is little quantitative information. Using model protein interactions with immobilized Cu2+ ions, we have compared analytical frontal affinity chromatographic methods to a simple, nonchromatographic protocol for the rapid determination of quantitative affinity relationships. Values obtained for the equilibrium dissociation constant (Kd) and binding capacity (Lt) characterizing the interaction of lysozyme with immobilized Cu2+ were quite similar by frontal analysis (Kd = 37-42 X 10(-6) M; Lt = 6.8-7.4 X 10(-6) mol protein/ml gel) and by equilibrium binding analyses (Kd = 33 +/- 4.7 X 10(-6) M; Lt = 5.8-6.1 X 10(-6) mol protein/ml gel; 14 determinations). The interaction of ovalbumin with immobilized Cu2+ was characterized by an affinity (Kd = 4.2-4.8 X 10(-6) M) and capacity (Lt = 1.5-2.1 X 10(-6) mol protein/ml gel) which were also the same regardless of the method for affinity analysis. These values indicate that the total protein bound at saturation corresponds to as much as 17% of the total immobilized Cu2+ ions (approximately 40 X 10(-6) mol/ml gel). Thus, depending on the fraction of total immobilized Cu2+ available for interaction with a given protein (e.g., lysozyme), the number of individual immobilized ligands actively participating as well as those rendered unavailable upon individual protein binding events may be greater than 1. Linear Scatchard plots obtained for both lysozyme and ovalbumin (purified) suggest the presence of only a single type of immobilized Cu2+-protein interaction operative under the experimental conditions employed. However, Scatchard analyses of data obtained by the nonchromatographic equilibrium binding method also demonstrated the ability to simultaneously resolve the contribution of two components whose presence was predicted by frontal chromatography. Our results support the validity and utility of equilibrium binding data analyzed according to the equations outlined by Scatchard and others as an alternative to analytical chromatographic methods.     

Interstitial cell heterogeneity in rat testes. I. Purification of collagenase-dispersed Leydig cells by unit gravity sedimentation and demonstration of binding sites for gonadotropin in light cells versus enhanced steroidogenesis in heavier cells

Two testicular interstitial cell fractions, light and heavier, biochemically and morphologically distinct were obtained by a unit gravity sedimentation procedure. Binding sites for 125I-labeled human chorionic gonadotropin (hCG) were preferentially localized in the light cell fraction (apparent Kd = 2.02 X 10(-10) M; Bmax = 1.17 X 10(-5) nmol/2 X 10(6) cells). These cells did not synthesize testosterone in response to hCG, but the basal release of testosterone was higher than by cells in the heavier fraction (2.49 +/- 0.02 ng/2 X 10(6) cells in the light versus 0.22 +/- 0.00 ng/2 X 10(6) cells in the heavier fraction). The cells in the heavier fraction bound little or no hCG. The binding data from this fraction did not obey saturation kinetics, but testosterone levels were elevated 700-800% in the presence of hCG (i.e. basal value 0.22 +/- 0.00 ng/2 X 10(6) cells versus 1.81 +/- 0.04 ng/2 X 10(6) cells in hCG-stimulated cells). Electron microscopy revealed that heavier cells had features typical of Leydig cells such as large ovoid nucleus with peripherally located heterochromatin, numerous mitochondria with tubular cristae, some lipid droplets, extensively developed smooth endoplasmic reticulum, and well developed Golgi complex. The cells in the light fraction contained an ovoid nucleus with one or more deep infoldings, and their most notable cytoplasmic feature was the presence of numerous vacuoles of varying sizes and shapes. Based upon this and the investigation which follows (Bhalla, V.K., Flasch, M.V., Browne, E.S., Sohal, G.S., and Sharawy, M.M. (1987) J. Biol. Chem. 262, 5322-5332), we conclude that occupancy of high affinity hCG binding sites, generally assumed to be coupled to steroidogenesis, is not necessarily related to the elicitation of this biological response.     

Bovine oviductal fluid components and their potential role in sperm cholesterol efflux

Bovine oviductal fluid (OF) was collected and analyzed throughout the estrous cycle, and the capacity of the protein and lipoprotein components to support cholesterol efflux from bovine sperm was evaluated. Blood was collected and assayed for progesterone (P4) to monitor the estrous cycle. Protein and lipoprotein separation was achieved by density gradient centrifugation. Two major bands were identified. The first (1.056 less than delta 20 less than 1.140 g/ml) corresponded to bovine and rabbit plasma high-density lipoprotein (HDL) based on distribution in the density gradient and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The second band (1.235 less than delta 20 less than 1.243 g/ml) consisted predominantly of oviductal fluid albumin (OFA). Oviductal fluid protein concentration increased as serum P4 decreased around the time of estrus. Mean OF protein concentration was 21.3 mg/ml when serum P4 was lower than 0.5 ng/ml and 6.9 mg/ml when serum P4 was greater than 0.5 ng/ml. An inverse log relationship was found between HDL protein concentration and serum P4. Unesterified cholesterol (UC), cholesteryl ester, and phospholipid (PL) content of HDL for HDL protein concentrations of 3-56.1 micrograms/ml were 1.35-46.2 micrograms/ml, 1.91-44.48 micrograms/ml, and 1.69-59.8 micrograms/ml, respectively. Phosphatidylcholine and -ethanolamine were the major PLs present in the HDL fraction and their molar ratio (4:1 mol/mol) was relatively constant through the estrous cycle. The OFA fraction of the same samples accounted for more than 90% of total protein and for most of the variation in OF protein. To determine the ability of OF components to serve as sperm cholesterol acceptors, OF samples were incubated 1:1 (v/v) with and without 4 X 10(8) bovine sperm in 1.0 ml of modified Tyrode's solution and OF for 2 hr at 39 degrees C. After incubation, HDL and OFA fractions were isolated and analyzed for changes in protein and lipid content. After OF, samples were incubated with sperm, an increase in UC was found in the HDL fractions. UC in HDL increased by 12.1 +/- 1.0 micrograms/ml (means +/- SE) when serum P4 was less than or equal to 0.5 ng/ml. For samples corresponding to higher serum P4, the increase in UC was 3.60 +/- 0.89 micrograms/ml. Values for UC in HDL were corrected for the contribution of UC from OFA of OF samples. Cholesterol efflux from sperm has been implicated in the process of sperm capacitation. These results indicate that HDL from OF is elevated during the follicular phase of the estrous cycle and can serve as an acceptor for bovine sperm cholesterol.     

High affinity epidermal growth factor receptors on the surface of A431 cells have restricted lateral diffusion

Rhodamine-labelled epidermal growth factor (Rh-EGF) was shown to bind to A431 cells grown at low density both to a small number of high affinity receptors (KD = 2.8 X 10(-10) M; fraction of total binding sites approximately 0.12) and also to a large number of low affinity receptors (KD = 4 X 10(-9) M; fraction of total binding sites approximately 0.88). Measurements of the lateral diffusion of EGF receptors on the cell surface were made using Rh-EGF and the technique of fluorescence photobleaching recovery. The high affinity receptors (labelled with 1.6 X 10(-10) M Rh-EGF, 5% of EGF binding sites occupied) did not show lateral mobility over the temperature range 3 degrees-37 degrees C. The low affinity receptors (labelled with 2.4 X 10(-7) M Rh-EGF, 90% of EGF sites occupied) showed at least 75% fluorescence recovery after photobleaching, and lateral diffusion coefficients of approximately 2 X 10(-10) cm2/s. These results show that the two populations of EGF receptors defined by binding studies differ in their freedom to diffuse laterally. The observation that the high affinity receptors are immobile indicates that lateral diffusion of receptors, at least over a distance of a few hundred nanometres or more, may not be required for the action of low concentrations of EGF.