Detectable Binding Activity Research Articles

Biosynthesis and processing of the type II insulin-like growth factor receptor in H-35 hepatoma cells.

The biosynthesis and post-translational processing of the insulin-like growth factor II (IGF-II) receptor has been studied in H-35 hepatoma cells using a specific polyclonal anti-receptor immunoglobulin preparation. Cells were pulse-labeled with [35S]methionine followed by incubation with excess unlabeled methionine (chase). Gel electrophoresis of the immunoadsorbed receptors shows that the receptor is first synthesized as a 245-kDa precursor which is transformed to the mature 250-kDa form with a half-time of about 2 h. The 245-kDa precursor could also be labeled biosynthetically with [3H]mannose, only one-half of which was ultimately found associated with the 250-kDa product. Neuraminidase converts the 250-kDa receptor species to a 245-kDa form. Whereas the 250-kDa receptor is insensitive to detectable cleavage by endoglycosidase H, digestion of the 245-kDa species with this enzyme produces a 232-kDa form. A similar 232-kDa receptor species accumulates in H-35 cells incubated with tunicamycin (2 micrograms/ml). This tunicamycin-induced aglyco-receptor is not further processed to the 250-kDa form. Monensin (50 nM) blocks receptor processing at the 245-kDa stage. Endoglycosidase H treatment of the monensin-induced 245-kDa species indicates that this is a mixture of partially processed precursors having equivalent Mr. No evidence was obtained for the presence of O-linked oligosaccharides on the IGF-II receptor. The IGF-II binding activity of the three different biosynthetic forms of the receptor was assessed by affinity cross-linking of 125I-IGF-II to the receptors using disuccinimidyl suberate. Both the mature 250-kDa receptor and the neuraminidase-digested 245-kDa form specifically bound 125-I-IGF-II. However, the 232-kDa aglyco-receptor had no detectable IGF-II binding activity using this method. In summary, these studies show: 1) that the H-35 cell IGF-II receptor is synthesized first as a 245-kDa precursor having 4-6 high-mannose oligosaccharide side chains, 2) processing of the receptor oligosaccharides by mannose removal and terminal sialylation converts the 245-kDa precursor to the 250-kDa mature product which has been previously identified as the functional receptor in the plasma membrane, 3) the apparent molecular mass of the receptor in the absence of N-glycosylation is 232-kDa, and 4) glycosylation of the IGF-II receptor is required for the acquisition of IGF-II binding activity.

Action of androgen on fibroblast collagen synthesis: a receptor-dependent response.

Several groups are still investigating an vitro biological response of androgen action. The purpose of this study was to determine whether dihydrotestosterone (DHT) influences the secretion of collagen by cultured foreskin fibroblasts raised from patients with normal or undetectable DHT-binding activity (complete androgen resistance). Collagen synthesis and secretion were evaluated by 3H-proline incorporation in newly synthetized collagen by confluent fibroblasts alone, or in the presence of DHT (10−5 to -10−10M). Production of collagen is expressed as a ratio between tritiated OH-proline and OH-proline plus proline,within the cells or in the culture medium. These data show that : -in control cells,DHT significantly increases production of collagen released in the culture medium (p < 0.001), -In complete androgen resistance with no detectable androgen binding activity, DHT does not modify basal collagen synthesis. These results strongly suggest that this hormonal response is mediated via a DHT-receptor or is receptor-dependent. This could be used as a biological response of androgen action in patients with androgen resistance associated with a post-receptor defect.

Isolation and Some Properties of a New Binding Protein for Asialoorosomucoid from Rat Liver&lt;xref ref-type="fn" rid="fn1"&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;

Binding proteins for asialoorosomucoid were prepared from rat liver previously labeled in vivo with [3H]leucine by affinity chromatography on asialoorosomucoid-Sepharose 4B. They were subjected again to the same affinity chromatography and eluted into two fractions successively with 10 mM Tris-HCl buffer, pH 7.8, containing 1.25 M NaCl, 1% Triton X-100 and 50 mM lactose and 20 mM ammonium acetate buffer, pH 6.0, containing 1.25 M NaCl and 1% Triton X-100, and designated as ABP-I and ABP-II (asialoorosomucoid binding proteins), respectively. ABP-I corresponds to the receptor protein specific for asialoglycoproteins which has been extensively investigated by Ashwell and collaborators (J. Biol. Chem. 254, 1038-1043, 1979). ABP-II is different from ABP-I in several properties such as molecular weight, antigenicity and solubility. The molecular weight of ABP-II was estimated to be 29,000 by SDS-PAGE. On gel filtration it behaved as a pentamer with an apparent molecular weight of 150,000. Unlike ABP-I, ABP-II showed no detectable binding activity when assayed according to the procedures of Hudgin et al. (J. Biol. Chem. 249, 5536-5543, 1974). The calcium ion was, however, essential for the binding of ABP-II to asialoorosomucoid-Sepharose 4B similar to ABP-I. ABP-II can be extracted from the total microsomes of rat liver in 1.0 M NaCl by sonication after freezing and thawing. This suggests that ABP-II is either a soluble protein or a peripheral membrane protein loosely attached to the intracisternal cavities of the microsomal membranes.

DNA-binding vesicles released from the surface of a competence-deficient mutant of Haemophilus influenzae

Haemophilus influenzae com-51, a mutant deficient in DNA uptake, produces an extracellular DNA-binding activity. The activity was specific for Haemophilus DNA and was isolated from cell-free competence medium after incubation for 100 to 130 min. Initial steps in the purification procedure resulted in the loss of detectable binding activity, but activity was restored by the addition of a nonionic detergent. The active fractions contained vesicles derived from the outer membrane of the cells. The vesicles were produced only under conditions that normally lead to competence development. The lack of competence of com-51 cells was not due to loss of protein synthesis in M-IV competence medium or to competition of extracellular protein for exogenous DNA. Results suggest that the inability of cells to bind DNA was due in part to the loss of DNA receptors that are released into the medium in membrane fragments.

Induction of idiotype-bearing, nuclease-specific helper T cells by in vivo treatment with anti-idiotype.

Treatment of BALB/c mice with purified pig anti-(BALB/c anti-nuclease) anti-idiotypic antibodies has been found to induce the appearance of idiotype-bearing immunoglobulins (Id') in the serum of these mice in the absence of detectable antigen binding activity. This phenomenon appeared to require T cells in the hosts because no Id' was detected in the serum of nude mice similarly treated. Furthermore, the spleens of BALB/c mice treated with anti-idiotype were found to contain helper T cells capable of providing help in an in vitro plaque-forming cell response to trinitrophenyl-nuclease equivalent to that provided by helper T cells from the spleens of nuclease-primed animals. Helper T cells from both anti-idiotype-treated and nuclease-treated animals were found to be antigen-specific and to be similarly susceptible to elimination by treatment with anti-idiotype plus complement. Therefore, treatment with both antigen and anti-idiotype appeared to prime similar populations of antigen-specific helper T cells, while having different effects on the induction of antibody. These findings are consistent with the network theory of receptor interactions in the immune response, and may provide a means for studying individual cell populations involved in such interactions.

Purification and Properties of a Periplasmic Protein

Protein GLPT, a periplasmic protein previously recognized as closely related to the active transport of sn-glycerol-3-phosphate in Escherichia coli was isolated by the cold osmotic shock procedure. It was purified by Sephadex chromatography and isoelectric focussing. The purified protein does not exhibit any detectable binding activity toward sn-glycerol-3-phosphate. It has no activity as a glycerol phosphatase nor as a glycerol kinase. Polyacrylamide gel electrophoresis in the presence of dodecylsulfate of the protein subsequent to treatment in urea, boiling in dodecylsulfate and crosslinking indicates that it occurs as an oligomeric protein composed of four identical subunits of 40 000 molecular weight. Membrane vesicles of wild-type strains that contain protein GLPT in whole cells loose it during vesicle preparation. However, they still exhibit high transport activity toward sn-glycerol-3-phosphate. Membrane vesicles prepared from glp T mutants that may or may not contain protein GLPT do not transport sn-glycerol-3-phospahte. We conclude from these results that protein GLPT does not participate in the energy-dependent active transport through the cytoplasmic membrane but could be involved in facilitating the diffusion of sn-glycerol-3-phosphate through the outer layers of E. coli.

The effect of vitamin D 3 metabolites on membrane proteins of chick duodenal brush borders

Chick intestinal brush border proteins were examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. Following injection of 25-hydroxyvitamin D 3 and 1,25-dihydroxyvitamin D 3, a large molecular weight protein present in the vitamin D-deficient brush borders diminishes and a larger protein appears. This change occurs before calcium binding protein can be detected by Chelex assay and prior to the increase in total alkaline phosphatase but correlates closely with increased intestinal calcium absorption in response to the metabolites. The two brush border proteins have been solubilized with n-butanol and partially characterized. The vitamin D-deficient protein has a molecular weight of about 200,000 and has alkaline phosphatase activity but no detectable calcium binding activity. The protein which appears in response to metabolites has a molecular weight of 230,000, binds calcium, and also has alkaline phosphatase activity.

X-chromosome linked inheritance of thyroxine-binding globulin deficiency.

ABSTRACT We have presented evidence for the inheritance of absent or reduced thyroxine binding globulin (TBG) capacity as a co-dominant, sex-linked trait (J Clin Endocr 26: 835, 1966). The study of a second unrelated family with this defect has yielded additional evidence that the trait is transmitted by x-chromosome linked dominant inheritance. The 8 male members of the present 3-generation kindred had no detectable TBG binding activity for either thyroxine or triiodothyronine. The 13 presumably heterozygous females had binding activity varying from very low to normal but always present. This pedigree contains normal male children of affected fathers, not demonstrated in the previous study. Thyroxine half-life studies in affected males gave times of 3.8 days as compared to the normal of 6–8 days. However, the daily thyroxine degradation rate was normal. Estrogen treatment of affected males did not elicit detectable TBG activity, whereas heterozygous females responded to treatment with increases in activi...

X-chromosome linked familial decrease in thyroxine-binding globulin activity.

Absent or reduced thyroxinebinding globulin activity has been found in 3 generations of a family. Ten male members of the kindred had no detectable binding activity, whereas 8 females, heterozygous for the trait, had binding activity varying from very low to normal, but always present. The character of the deficit and the mode of transmission of the trait are consistent with a sex-linked or X-chromosome linked dominant inheritance. The affected males had low protein-bound iodine levels (mean = 2.1 μg/100 ml) and high resin T3 uptake levels (mean =61%). Serum cholesterol levels were normal (mean = 196 mg/100 ml) and the individuals appeared clinically euthyroid. Other serum proteins, including total protein, albumin, thyroxine-binding prealbumin, corticosteroidbinding globulin, ceruloplasmin, haptoglobin and transferrin were normal. The response of these proteins to estrogen treatment was normal in an affected male, but TBG activity remained undetectable. The heterozygous females presented a more variable ...