Plasma clearance and liver metabolism of native and asialo-human transcortin in the rat

Plasma kinetics and liver metabolism of iodanated human corticosteroid-binding protein have been studied in ovariectomized female rats. ¹²⁵I-labeled human corticosteroid-binding globulin prepared by a modified chloramine T reaction was shown to be physically intact and biologically active. Intravenously injected ¹²⁵I-labeled human corticosteroid-binding globulin was shown to give a complex clearance pattern from the plasma, with half-lives of 7.5 and 51 min. Estrogen injections had no effect on plasma clearance rate. Direct involvement of liver plasma membrane receptors for asialoglycoproteins in ¹²⁵I-labeled human corticosteroid-binding globulin metabolism was demonstrated in vivo and in vitro using asialofetuin as a competitive inhibitor. ¹²⁵I-labeled human asialo-corticosteroid-binding globulin was cleared from the plasma with a half-life of less than 1 min, while the simultaneous injection of 5 mg asialofetuin maintained the circulating plasma levels. Asialofetuin also slowed the clearance of intact ¹²⁵I-labeled human corticosteroid-binding globulin from the plasma ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=90}\text{min}$). Binding of ¹²⁵I-labeled human asialo-corticosteroid-binding globulin to rat liver plasma membranes in vitro was inhibited in a dose-dependent manner by asialofetuin, but not by intact human corticosteroid-binding globulin or fetuin. ¹²⁵I-labeled human corticosteroid-binding globulin did not bind significantly to the membranes. It is concluded that human corticosteroid-binding globulin clearance from rat plasma is rapid and that the carbohydrate moiety of human corticosteroid-binding globulin is involved in its clearance and catabolism by the liver.     

Stimulation of ornithine decarboxylase synthesis in the rat thyroid

High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme·monospecific antibody complex as the immunizing antigen. This new antiserum preparation was successfully labeled with ¹²⁵I and was found to retain its specific immune properties. Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone and methyl xanthine in rat thyroids $\text{in vitro}$. ¹²⁵I-labeled antibody incorporation into the enzyme antibody complex after induction $\text{in vitro}$ showed an increase which paralleled the increase in enzymatic activity and thus suggested $\text{de novo}$ synthesis of thyroid enzyme protein.     

Production and characterization of an antiserum to synthetic gonadotropin-releasing hormone

The synthetic decapeptide “luteinizing hormone-releasing hormone” (LH-RH) was rendered antigenic by reaction of its histidine or tyrosine residues (7 : 3 approx.) with p-diazonium phenylacetic acid and coupling of the azo-derivatives formed to bovine serum albumin (BSA). Immunization of rabbits yielded antisera that bound ¹²⁵I-labeled LH-RH (approx. 50 pg) at dilutions up to 1:200, 000 and showed no cross-reaction with unrelated hypothalamic and pituitary hormones, extracts from rat cerebral cortex, and with small fragments of LH-RH. Cross-reaction was minimal (0.2%) with the free acid analogue of LH-RH, and moderate with $\text{des}$-pGlu LH-RH (20%), $\text{des}$-pGlu-His-LH-RH (2.4%) and with LH-RH analogues in which a single residue (No. 4–6 or No. 8) was exchanged by an amino-acid of similar character (1.2–12%). Biologically active hypothalamic extract and LH-RH produced parallel ¹²⁵I-LH-RH-binding inhibition curves, providing immunochemical support for the identity of the native releasing hormone with synthetic LH-RH.     

Saturable binding to cell membranes of the presynanptic neurotoxin, β-Bungarotoxin

Brief exposure to the protein neurotoxin, β-bungarotoxin, is known to disrupt neuromuscular transmission irreversibly by blocking the release of transmitter from the nerve terminal. This neurotoxin also has a phospholipase A2 activity, although phospholipases in general are not very toxic. To determine if the toxicity of this molecule might result from specific binding to neural tissue, we have looked for high affinity, saturable binding using ¹²⁵I-labeled toxin. At low membrane protein concentration ¹²⁵I-labeled toxin binding was directly proportional to the amount of membrane; at fixed membrane concentration ¹²⁵I-labeled toxin showed saturable binding. It was unlikely that iodination markedly changed the toxin's properties since the iodinated toxin had a comparable binding affinity to that of native toxin as judged by competition experiments. Comparison of toxin binding to brain, liver and red blood cell membranes showed that all had high affinity binding sites with dissociation constants between one and two nanomolar. This is comparable to the concentrations previously shown to inhibit mitochondrial function. However, the density of these sites showed marked variation such that the density of sites was 13.0 pmol/mg protein for a brain membrane preparation, 2.4 pmol/mg for liver and 0.25 pmol/mg for red blood cell membranes.In earlier work we had shown that calcium uptake by brain mitochondria is inhibited at much lower toxin concentrations than is liver mitochondrial uptake. Both liver and brain mitochondria bind toxin specifically, but the density of ¹²⁵I-labeled toxin binding sites on brain mitochondrial prepartions ($\text{3.3 ± 0.3}\text{pmol/mg}$) exceeded by a factor of ten the density on liver mitochondrial preparations ($\text{0.3 ± 0.05}\text{pmol/mg}$). It is also shown that the labeled toxin does not cross synaptosomal membranes, suggesting that mitochondria may not be the site of action of the toxin in vivo. We conclude the β-bungarotoxin is an enzyme which can bind specifically with high affinity to cell membranes.     

Identification of albumin-synthesizing polysomes from mouse liver and a mouse hepatoma cell line.

Albumin-synthesizing polysomes from mouse liver and mouse hepatoma cells in in tissue culture have been localized on sucrose gradients with ¹²⁵I-labeled antimouse serum albumin used as a marker. Competition studies show that the ¹²⁵I-labeled antibody binds specifically to albumin-synthesizing polysomes from both tissues. The ¹²⁵I-labeled polysomes from liver and hepatoma cells have identical sedimentation properties on sucrose gradients, which indicates that the polysomes range in size from 9–14 ribosomes. This is comparable in size to polysomes from rat liver and Morris hepatoma. One significant difference between these albumin-synthesizing polysomes is that those extracted from hepatoma cells bind 70% less antibody than equivalent amounts of polysomes from liver cells. Since the level of albumin synthesis in the hepatoma cells is comparable to the level of albumin synthesis $\text{in vivo}$, this difference in antibody-binding capacity is not likely to be due to differences in polysomal content, but appears to be a characteristic difference between hepatoma and normal mouse liver cells.     

Lectin and cholera toxin binding to guinea pig tumor (104C1) cell surfaces before and after glycosphingolipid incorporation

¹²⁵I-Labeled $\text{Dolichos biflorus}$ lectin and cholera toxin were used as probes for identification of Forssman- and GM1-type receptor sites on guinea pig tumor (104C1) cell surfaces. Increased binding of ¹²⁵I-labeled lectin and toxin to 104C1 cell surfaces was observed after the cells were treated with exogenous Forssman glycosphingolipid and GM1 ganglioside, respectively. Biosynthesis $\text{in vitro}$ of these two glycosphingolipids from their precursor molecules was established using a membrane preparation isolated from confluent cultures of guinea pig tumor 104C1 cells.     

Biosynthesis in vitro and the pattern of lectin binding receptors in monkey kidney cell surfaces.

The glycosyltransferase activities involved in the biosynthesis $\text{in vitro}$ of neutral blood group-related glycosphingolipids were measured in African green monkey kidney cells (Vero) grown in culture. The a-fucosyltransferases which catalyzed the reaction between GDP-fucose and corresponding acceptors to form H-active and novel Le^a-type glycosphingolipids were characterized in membrane fractions isolated from Vero cells and monkey bone marrow. Using ¹²⁵I-labeled $\text{Ulex europeus}$ and $\text{Lotus tetragonolobus}$ lectins the differential binding to Vero cell surface glycoproteins and glycolipids was studied under various conditions.     

Identification of immunogens of Mycoplasmapneumoniae by protein blotting

Proteins of $\text{Mycoplasma}\text{pneumoniae}$ were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose sheet by blotting. Sera obtained from infected hamsters and immunized rabbits were then incubated with the nitrocellulose strips. Proteins which are capable of eliciting antibodies were detected by indirect immunoradioautography using ¹²⁵I-labeled antisera against hamster or rabbit IgG. Antibodies to seven immunogens were demonstrated in the sera of hamsters infected with $\text{M.}\text{pneumoniae}$ by inhalation, while many more proteins were found to be capable of stimulating antibodies in rabbits immunized parenterally with mycoplasmas. This method should prove useful for identifying those components of a microorganism which elicit antibody responses and contribute to the protective state.     

Development of the neuromuscular junction in the chick embryo: The number,distribution, and stability of acetylcholine receptors

The number, distribution, and stability of skeletal muscle acetylcholine receptors during development of the neuromuscular junction in the chick embryo were studied. The distribution and turnover of receptors labeled with ¹²⁵I-labeled α-bungarotoxin were determined by quantitative autoradiography on individual teased muscle fibers. Each posterior latissimus dorsi muscle fiber, which in the adult is singly innervated, has a high density of acetylcholine receptors at a single spot from embryonic Day 10 through hatching. The spots stain more intensely than elsewhere for acetylcholinesterase and are assumed to be end plates. The receptors at these spots are presumed to be junctional receptors. The junctional receptor density remains constant at 10⁴/μm² from embryonic Day 14 through adult life, although the area of the junction increases 40-fold. In contrast, the extrajunctional receptor density drops precipitously from 250/μm² on Day 14 to only 10/μm² on Day 19. This decrease in extrajunctional receptor density can be prevented by chronic paralysis with curare. The rate of autoradiographic grain loss from junctional and extrajunctional regions after a pulse injection of ¹²⁵I-labeled α-bungarotoxin indicates that both classes of embryonic receptors turn over at the same rate ($\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 30}\text{hr}$).     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.     

Insulin binding in cultured Chinese hamster kidney epithelial cells the effects of glucose concentration in the medium and tunicamycin

An epithelial cell line established from a Chinese hamster kidney, CHK-AC_E, was separated into two sublines, CHK-AC_E-100 and CHK-AC_E-400, by 18 successive passages in medium containing 100 and 400 mg/dl glucose, respectively. Binding of CHK-AC_E-100 and CHK-AC_E-400 cells to ¹²⁵I-labeled insulin showed similar pH and time dependency; ¹²⁵I-labeled insulin binding as a function of insulin concentration differed in the two sublines, however. Degradation of ¹²⁵I-labeled insulin, as determined by its ability to bind insulin antibody and cells, was more extensive when preincubated with CHK_AC_E-400 cells than with CHK-AC_E-100 cells. When CHK-AC_E-100 cells were grown in 400 mg/dl glucose for six passages, these cells showed more insulin binding sites than cells grown parallel in 100 mg/dl glucose; whereas CHK-AC_E-400 cells grown in 100 mg/dl glucose for six passages showed fewer insulin binding sites than those grown parallel in 400 mg/dl glucose. A slight increase in $\text{K}{}_{\mathrm{<mtext>f</mtext>}}\text{/}\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ ratio was observed in both sublines when grown in 400 mg/dl glucose as compared to 100 mg/dl glucose, indicating attenuated negative cooperativity of the binding sites in cells grown in 400 mg/dl glucose. Tunicamycin, at concentrations from 0.016 to 0.125 μg/ml, showed no direct effect on the assay of ¹²⁵I-labeled insulin binding to CHK-AC_E-100 cells; exposure of CHK-AC_E-100 cells to tunicamycin, at concentrations from 0.01 to 0.2 μg/ml, for 24 h caused a dose-dependent decrease in insulin binding capacity and an increase in $\text{K}{}_{\mathrm{<mtext>f</mtext>}}\text{/}\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ ratio. These data indicate that the number of insulin binding sites in the cultured Chinese hamster kidney epithelial cells increased with high glucose concentrations in the culture medium, whereas tunicamycin, an inhibitor of protein glycosylation, lowered the number of insulin binding sites.     

Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells.

The protein neurotoxin II from the venom of the scorpion $\text{Androctonus}\text{australis}$ Hector was labeled with ¹²⁵I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non intereacting binding sites of high affinity (K_D = 0.3 – 0.6 nM) and low capacity (4000 – 8000 sites/cell) to electrically excitable neuroblastoma cells. Relation of these sites to the action potential Na⁺ channel is derived from identical concentration dependence of scorpion toxin binding and increase in duration and amplitude of action potential. The protein neurotoxin II from the sea anemone $\text{Anemona sulcata}$ also affects the closing of the action potential Na⁺ ionophore in nerve axons. The unlabelled sea anemone toxin modifies ¹²⁵I-labeled scorpion toxin II binding to neuroblastoma cells by increasing the apparent K_D for labeled scorpion toxin without modification of the number of binding sites. It is concluded that both $\text{Androctonus}$ scorpion toxin II and $\text{Anemona}$ sea anemone toxin II interact competitively with a regulatory component of the action potential Na⁺ channel.     

Loose binding of testicular mitochondrial ATPase to the inner membrane

Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N′-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F₁ to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few “knobs” or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was $\text{30}\text{1}$ in liver mitochondria, whereas in the testis it was $\text{3}\text{1}$. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.     

Relationship between testosterone, fluid content and luteinizing hormone receptors in the rat testis.

Injection of immature male rats with human chorionic gonadotrophin resulted in a decreased ability of the testis to bind [¹²⁵I]-labelled human chorionic gonadotrophin $\text{in vitro}$, and a marked, but transient increase in testis weight; the latter was apparently due to the accumulation of fluid containing high levels of testosterone. Intra-testicular injection of cycloheximide significantly inhibited all these changes, thus demonstrating their dependence on protein synthesis. It is concluded from this and other data that either testosterone itself or a steroidogenic protein intermediary may be responsible for the gonadotrophin-induced reduction in availability of gonadotrophin receptors.     

Peptide receptors in human lung tumor cells in culture: vasoactive intestinal peptide (VIP) and secretin interaction with the Calu-1 and SW-900 cell lines

We have investigated the interaction of VIP and secretin with two human lung carcinoma cell lines in cultures, SW-900 and Calu-1. ¹²⁵I-labeled VIP binds to and is inactivated by SW-900 and Calu-1 cells in a time- and temperature-dependent manner. The rates of binding and of inactivation were higher at 30°C than at 15°C. At equilibrium, native VIP competitively inhibited the binding of ¹²⁵I-VIP in the 10^?10?10^?7M range, half-maximal inhibition being observed at 1.2 nM in SW-900 cells and at 1.1 nM VIP in Calu-1 cells. Scatchard analysis indicated two classes of binding sites with similar characteristics in both cell lines. SW-900 cells have 27 600 sites with a high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 0.34}\text{nM}$) and 1062 000 sites with a low affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 61.4}\text{nM}$). Calu-1 cells have 36 300 sites with a high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 0.33}\text{nM}$) and 1148 000 sites with a low affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 78.6}\text{nM}$). Secretin inhibited tracer binding but with a 5000 times lower potency than native VIP in both cell lines.     

Erythroid specific nuclear antigen and globin gene binding protein

Using the procedure of Bekhor and Mirell (Biochem, $\text{18}$, 609, 1979), we isolated a nonhistone protein fraction tightly bound to DNA which putatively has a role in globin gene regulation in chicken reticulocytes. This fraction was tested by gel electrophoresis and microcomplement fixation and appears by these criteria very similar to the chicken nuclear antigen previously identified in reticulocyte chromatin and structurally altered erythrocyte chromatin. This antigen is tissue and species specific (Pumo $\text{et}\text{al}$, Biochem. $\text{19}$, 2362, 1980).     

Mitochondrial heterogeneity in foetal and suckling rat liver: differential leucine incorporation into the proteins of two mitochondrial populations.

Iodination of intact mitochondria with ¹²⁵I results in the labeling of essentially one polypeptide with an approximate MW of 30 000. This polypeptide seems to be a component of the inner boundary membrane as it can not be removed from the mitochondria by procedures which destroy the outer membrane (e.g. incubation with digitonin). The amount of the radio-active label which can be bound to this polypeptide is determined by ADP, atractylate, and bongkrekate, components which act on the functional state and the position of the $\text{ADP}\text{ATP}$ carrier in the membrane. [1,2]     

Association of α2-macroglobulin-thrombin and α2-macroglobulin-plasmin complexes with isolated hepatocytes

¹²⁵I-labelled $\text{α}{}_2\text{-}\text{macroglobulin}$ complexed with thrombin or plasmin bound to hepatocytes in a concentration-and time-dependent manner. The apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values calculated from displacement experiments were 7.9 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ and 8.5 · 10^?8 M for $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$. Association of these complexes was only partially reversible; after a 180 min incubation period, 50–60% of the bound radioactivity was internalized by the cells. $\text{α}{}_2\text{-}\text{Macroglobulin}$ itself bound also to hepatocytes, but the affinity of the $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes was higher than that of the inhibitor alone, and $\text{α}{}_2\text{-}\text{macroglobulin}$ was not internalized, either. ¹²⁵I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their $\text{α}{}_2\text{-}\text{macroglobulin}$ complexes. The $\text{α}{}_2\text{-}\text{macroglobulin-thrombin}$ complex competed with the $\text{α}{}_2\text{-}\text{macroglobulin-plasmin}$ complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for $\text{α}{}_2\text{-}\text{macroglobulin-proteinase}$ and antithrombin III-proteinase complexes are different.