Toxic ligand conjugates as tools in the study of receptor-ligand interactions

We have constructed hybrid proteins in which the toxic A chains of ricin or diptheria toxin have been linked to either asialofetuin, fetuin, or epidermal growth factor (EGF). Both ASF-RTA and ASF-DTA are potent toxins on cultured rat hepatocytes, cells that display the asialoglycoprotein receptor. Toxicity of these two compounds is restricted to hepatocytes and can be blocked by asialoglycoproteins but not the native glycoproteins or asialoagalactoglycoprotein derivatives, indicating that the toxicity of the conjugates is mediated by the hepatic asialoglycoprotein receptor. The EGF-RTA conjugate is an extremely potent toxin on cells that can bind the hormone, but is only poorly effective on cells that are unable to bind EGF. The EGF-DTA conjugate, in contrast, is unable to kill 3T3 cells and is at least two orders of magnitude less effective than EGF-RTA on A431 cells, a cell line with 1-2 X 10(6) EGF receptors per cell. However, when EGF-RTA and EGF-DTA were tested on primary liver hepatocyte cultures, which were susceptible to both ASF-RTA and ASF-DTA, both EGF conjugates were potent toxins. Sensitivity of the hepatocyte cultures to ricin toxicity increases slightly during a 52-hr culture period. In contrast, sensitivity to EGF-RTA and ASF-RTA decline dramatically during this period. Receptors for both ligands remain plentiful on the cell surface during this time.     

The Benzodiazepine/GABA Receptor Complex: Molecular Size in Brain Synaptic Membranes and in Solution

Abstract: The molecular size of the benzodiazepine (BZ) receptor in the synaptic membrane of brain cortex (bovine or rat) was determined by an improved version of the radiation inactivation method to be 220,000. An identical size was found simultaneously for the associated γ-aminobutyric acid (GABA) receptor and for the component binding β-carboline esters. It is proposed that all three activities reside in a single protein or protein complex in the membrane. The size in solution, after extraction into Triton X-100 medium from exhaustively washed membranes, was estimated by sedimentation constant (9.4S) and by gel filtration (∼230,000 apparent MW), again with the BZ and GABA binding activities behaving identically. This size applies to the component that undergoes photoaffinity labelling by [³H]flunitrazepam in the membrane, and contains a 51,000 M_r polypeptide as the BZ-binding subunit. It is concluded that a protein complex or oligomer of 200,000–220,000 MW carries a class of BZ-binding sites and an associated class of GABA_A sites.     

Multiple Molecular Forms of Acetylcholine Receptors in Cultured Skeletal Muscle Cells: Subcellular Localization and Characterization

Abstract: Skeletal muscle cells of newborn rats, cultured in the absence of neuronal influence, were found to contain two types of cell surface acetylcholine receptors as demonstrated by isoelectric focusing. The isoelectric points of the two types of receptors were indistinguishable from those of junctional and extrajunctional types of receptors in mature animals. The cultured cells had two classes of intracellular α-bungarotoxin (αBT) binding components; one had the same sedimentation coefficient as that of surface receptors (9S), and the other had much smaller apparent molecular weights. Only a single major component was detected by isoelectric focusing analysis of the 9s intracellular aBT binding component, with a PI value close to that of the extra junctional receptor. These results suggest that the junctional and extrajunctional types of receptors may be synthesized through a common precursor.     

Steroid hormone receptor studies in melanoma model systems

The Transplantable B-16 melanotic melanoma carried in syngeneic C57B1/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrosedensity gradient centrifugation of cytosol after incubation with [³H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5–35 fmoles per mg cytosol protein. Scatchard analysis of [³H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 × 10^?10 M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [³H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [³H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high affinity sites with a dissociation constant of 2 × 10^?9 M. Binding levels from 70–610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.     

Human liver aldehyde dehydrogenase in Chinese and Asiatic Indians: Gene deletion and its possible implications in alcohol metabolism

Two separate human liver aldehyde dehydrogenases exist which show differences in substrate specificity, cation inhibition or activation, and molecular weight. In this paper we report a common absence of enzyme 2 in Chinese which may be taken to indicate a gene deletion coding for this enzyme. The possible implication of this gene deletion among Chinese is discussed.     

Assignment of the sorbitol dehydrogenase locus to human chromosome 15 pter→q21

The locus for sorbitol dehydrogenase (SORD, E.C. 1.1.1.14) has been shown to segregate with hexosaminidase A and mannose phosphate isomerase in a series of human-Chinese hamster somatic cell hybrids. Cytogenetic analysis supports the assignment to chromosome 15 and further defines the gene locus to the region 15pterq21.This research was supported by the Medical Research Council of Canada (MT 4061), the Children's Hospital of Winnipeg Research Foundation, Inc., and the Department of Health, Province of Manitoba (H.S.W.).     

Genetics of formamidase-5 (brain formamidase) in the mouse: Localization of the structural gene on chromosome 14

A single formamidase, which is different from the formamidases found in other tissues, occurs in the brains of mice. This enzyme is here called formamidase-5 and the gene symbol is designated For-5. Two alleles are recognized on the basis of their differential heat sensitivity: For-5 ^b is relatively heat stable and is present in strain C57BL/6J, while For-5 ^d is relatively heat sensitive and is present in strain DBA/2J. The heat sensitivity of formamidase-5 in 44 other inbred strains and substrains was tested and found to resemble that of C57BL/6J or DBA/2J. Thirty-six recombinant inbred strains derived from progenitors that differed at For-5 were studied to test for single-gene inheritance and linkage with other loci. Complete concordance was found with the esterase-10 locus (Es-10), indicating close linkage. The 99% upper confidence limit of the distance between For-5 and Es-10 is 3.7 centimorgans (cM). Es-10 is located on chromosome 14 about 19 cM from the centromere. An independent demonstration of linkage of For-5 with Es-10 and another chromosome 14 marker, hairless (hr), is provided by the finding that the HRS/J strain, which has been sibmated for 60 generations with forced heterozygosity at the hr locus, is cosegregating at For-5 and Es-10. A survey of 32 inbred strains and substrains revealed that the For-5 ^d allele is associated with the Es-10 ^b allele, and that the For-5 ^b allele is associated with Es-10 ^a and Es-10 ^c. Formamidase-5 segregates as expected in the F₂ generation of crosses between strains bearing For-5 ^b and For-5 ^d alleles. It is possible that this unique formamidase of the brain is involved in the metabolism of a neurotransmitter substance.This research was sponsored in part by the Department of Energy under contract with the Union Carbide Corporation and in part by NIH Research Grant GM-18684 from the National Institute of General Medical Sciences. J. C. F. is a predoctoral Fellow supported by Grant CA 09104 from the National Cancer Institute. The Biology Division of Oak Ridge National Laboratory and the Jackson Laboratory are fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Evidence for the proximity of a cysteinyl and a tyrosyl residue in the active site of 6-phosphogluconate dehydrogenase

The treatment of 6-phosphogluconate dehydrogenase from Candida utilis with dansyl chloride causes the modification of one amino acid residue per enzyme subunit and the inactivation of the enzyme. Either a cysteine or a tyrosine residue can be modified, depending on the pH of the reaction mixture. The dansyl residue can be transferred from one residue to the other suggesting that the two amino acid residues are close in the tridimensional structure of the active site of the enzyme.     

Cell shape changes and transmembrane receptor uncoupling induced by tertiary amine local anesthetics

Tertiary amine local anesthetics (dibucaine, Tetracaine, procaine, etc.) modify cell morphology, concanavalin A (Con A)-mediated agglutinability and redistribution of Con A receptors. Con A agglutination of untransformed mouse 3T3 cells was enhanced at low concentrations of local anesthetics, and the dynamics of fluorescent-Con A indicated that ligand-induced clustering was increased in the presence of the drugs. In contast, these drugs inhibited Con A-induced receptor capping on mouse spleen cells. These effects can be duplicated by combinations of vinblastine (or colchicine) and cytochalasin B suggesting that local anesthetics act on microtubule cell surface receptor mobility and distribution. It is proposed that tertiary amine local anesthetics displace plasma membrane-bond Ca²⁺, resulting in disengagement of microfilament systems from the plasma membrane and increased cellular Ca²⁺ concentration to levels which disrupt microtubular organization. The possible involvement of cellular Ca²⁺ in cytoskeletal destruction by local anesthetics was investigated utilizing Ca²⁺-specific ionophores A23187 and X537A. In media containing Ca²⁺ and cytochalasin B these ionophores caused effects similar to tertiary amine local anesthetics.