Rat liver glutathione S-transferases. Construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit

Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B. S. (1983) Gene 25, 263-269). Recombinant clone, pGTB42, contained a cDNA insert of 900 base pairs whose 3' end showed specificity for the Yc mRNA in hybrid-select translation experiments. The nucleotide sequence of pGTB42 has been determined, and the complete amino acid sequence of a Yc subunit has been deduced. The cDNA clone contains an open reading frame of 663 nucleotides encoding a polypeptide comprising 221 amino acids with a molecular weight of 25,322. The NH2-terminal sequence deduced from pGTB42 is in agreement with the first 39 amino acids determined for a Ya-Yc heterodimer by conventional protein-sequencing techniques. A comparison of the nucleotide sequence of pGTB42 with the sequence of a Ya clone, pGTB38, described previously by our laboratory (Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J.-F., Argenbright, L., and Lu, A.Y.H. (1984) J. Biol. Chem. 259, 5182-5188) reveals a sequence homology of 66% over the same regions of both clones; however, the 5'- and 3'-untranslated regions of the Ya and Yc mRNAs are totally divergent in their sequences. The overall amino acid sequence homology between the Ya and Yc subunits is 68%, however, the NH2-terminal domain is more highly conserved than the middle or carboxyl-terminal domains. Our data suggest that the Ya and Yc subunits of the rat liver glutathione S-transferases are products of two different mRNAs which are derived from two related yet different genes.     

Kinetic evidence for differential agonist and antagonist binding to bovine hippocampal synaptic membrane opioid receptors

To examine the kinetics of opioid receptor binding, the agonists [D-Ala2-D-Leu5]enkephalin (DADL) and [D-Ala2-MePhe4-Gly-ol5]enkephalin (DAGO) and the antagonists diprenorphine and naltrexone were used with bovine hippocampal synaptic plasma membranes. By computer modeling of equilibrium binding displacement curves utilizing the LIGAND program, we found opioid peptides bind with high affinity to single populations of synaptic plasma membranes receptors, whereas opiate alkaloids bind to multiple sites. Initial kinetic experiments revealed that agonist rates of association were radioligand concentration-independent. Pseudo first-order rate constants for DADL, DAGO, diprenorphine, and naltrexone association were estimated to be 5.63 X 10(5), 5.08 X 10(5), 4.60 X 10(6), and 2.3 X 10(6) mol-1 X s-1, respectively. After preincubation of 0.2-1 nM radioligand for variable time intervals, dissociation was initiated by addition of 1 microM unlabeled ligand. If saturation binding was achieved before dissociation was initiated, then nearly monophasic dissociation of DADL, DAGO, and diprenorphine and a biphasic off-rate for naltrexone were observed. When association times were reduced to pre-equilibrium intervals, the kinetics of dissociation of agonists became biphasic and association time-dependent, but that for antagonists did not change significantly. Comparisons by both graphical methods and computerized nonlinear regression analyses of rate constants revealed that the fraction of the rapid component of agonist dissociation decreases and that of the slow component is elevated with increasing receptor occupancy. In the presence of 100 mM NaCl, DADL dissociation became association time-independent. These data are consistent with the idea that the Na+ effect is brought about by a change of receptor to an antagonist-like conformation. On the basis of both association and dissociation kinetic data, opioid agonists appear to interact in a multistep process in which a rapid, reversible association is followed by the formation of a more tightly bound complex.     

Molecular organization of paramyosin in the core of molluscan thick filaments

An arrangement of paramyosin molecules in the polar part of molluscan thick filaments is proposed which accounts for the X-ray diffraction pattern of the smooth adductor muscle (other than the part ascribed to actin) and for the appearance of separated filaments in the electron microscope. The proposed structure is based on the PI arrangement of Cohen et al. (1971), and contains sets of parallel, equidistant molecules with successive molecules displaced along the molecular axis by 72 nm, which we call PI sheets. Every molecule belongs to two PI sheets which are nearly perpendicular. This array is not propagated throughout the filament, but is sheared periodically in the direction of the molecular (filament) axis by 2/5 X 72 nm. The shear occurs along parallel equidistant planes which are inclined to the PI sheets. The analysis of the X-ray data has been made possible by concentrating on those patterns from filaments in which the two sets of PI sheets appear to be mutually perpendicular, a condition brought about by bathing the muscle in aqueous acetone. In one set, there are four intermolecular spaces between shear planes (this appears to be true at least for the smooth adductors of Ostrea edulis, Crassostrea angulata and Mercenaria mercenaria). In the other set, the number varies with species and probably lies between eight and ten in the first two and appears to be six in the last named species. The known paracrystalline nature of paramyosin filaments suggests that this number, though dominant in one species, is not exactly constant.     

Breast volume and anthropomorphic measurements: normal values

Fifty-five consecutive female volunteers were evaluated. Linear measurements of the breast and nipple position were determined to fixed points on the chest wall. Volume determination was done by a standard chest-wall casting technique. A highly significant difference was found between left and right breasts in the axilla-to-nipple distance and nipple-to-midline measurements. A significant difference was also found in the distance from the lowest point of the breast (patient upright) to the nipple. Analysis of the volumetric difference between right and left breasts did not achieve statistical significance.     

Interaction of AP-2, a monoclonal antibody specific for the human platelet glycoprotein IIb-IIIa complex, with intact platelets

A murine monoclonal antibody, designated AP-2, reacts specifically with the complex formed by human platelet membrane glycoproteins IIb and IIIa, but does not react at all with the individual glycoproteins. Purified AP-2 covalently coupled to Sepharose CL4B was used as an immunoadsorbent column to purify the IIb-IIIa complex from a preparation of Triton X-100-solubilized human platelet proteins. Radioiodinated AP-2 was shown to bind to a single class of sites, with 57,400 +/- 9,700 molecules bound per cell (mean +/- S.D.) at saturation and a dissociation constant (Kd) of 0.64 +/- 0.15 nM (mean +/- S.D.). Binding could not be readily reversed even after a 1-h incubation with a 100-fold excess of cold antibody. AP-2 inhibits ADP-induced binding of radiolabeled fibrinogen to gel-filtered platelets in a noncompetitive fashion, consistent with the previous observation that AP-2 also inhibits the aggregation of platelets in plasma induced by a number of physiologic agonists, including adenosine diphosphate, epinephrine, collagen, thrombin, and arachidonic acid. Using AP-2, we have obtained evidence that the IIb-IIIa complex exists in the membrane of intact nonstimulated platelets and that complex integrity is not affected by external calcium ion concentration.     

alpha-N-acetyl-beta-endorphin1-26 from the neurointermediary lobe of the rat pituitary: isolation, purification, and characterization by high-performance liquid chromatography

The neurointermediary lobes from 190 rat pituitaries were homogenized in an acidic medium which inhibits peptidase activity and maximizes the solubilization of undamaged peptides. Octadecylsilyl-silica (ODS-silica) was used to extract the supernatant of the tissue homogenate. The ODS-silica eluate, now largely protein and salt free, was subjected to reversed-phase high-performance liquid chromatography (HPLC) employing 0.1% trifluoroacetic as counter ion. The column eluates were monitored for beta-endorphin immunoreactivity. Five immunoreactive components were observed. The most hydrophobic of these was repurified on the same HPLC column using 0.13% heptafluorobutyric acid as counter ion. Characterization of the purified peptide by gel permeation HPLC, amino acid analysis, and tryptic fragmentation indicated that it corresponded in structure to alpha-N-acetyl-beta-endorphin1-26. Amino acid analysis of the native peptide and its trypsin and carboxypeptidase fragments indicated that an alanyl residue occupies position 26. This finding is in contrast to the sequence predicted for the beta-lipotropin/corticotropin precursor by recombinant DNA techniques which suggests that the 26th residue of the beta-endorphin molecule should be valine.     

Human phosphoserine 31 corticotropin1-39. Isolation and characterization

Two distinct forms of corticotropin1-39 (ACTH) were isolated and purified from an extract of three adult human pituitaries by reversed-phase chromatographic techniques. Structural studies indicated that the more polar form of ACTH was phosphorylated at serine residue 31. Approximately 30% of the ACTH was found in the phosphorylated form. A similar proportion of phosphorylated ACTH was observed in extracts of three pituitaries from human fetuses of 15, 17, and 20 weeks gestation. Phosphorylated and nonphosphorylated human ACTH1-39 were found to be steroidogenically equipotent using both an isolated rat adrenal cell bioassay and a cultured human fetal adrenal cell bioassay.     

Carbohydrate changes in pre- and peri-implantation mouse embryos as detected by a monoclonal antibody

We have examined the tissue and embryonic distribution of an antigen on a large polysaccharide that is recognized by a monoclonal antibody, IIC3, prepared against F9 teratocarcinoma cells. By immunofluorescence the antigen is first detected on compacted morulae and early blastocysts. It is strongly expressed on the primary endoderm and trophoblast of expanded blastocysts, but then disappears from the trophoblast of attached blastocysts in vitro. The binding of the antibody is completely inhibited by D-galactose and N-acetylgalactosamine. Fluoresceinated lectins were used to study further the changes in cell surface carbohydrates on trophoblast during implantation. Ricinus I, specific for terminal galactose, binds to preimplantation stages but does not bind to the trophoblast of the attached blastocyst. On the other hand, wheat germ agglutinin, specific for N-acetylglucosamine and sialic acid, binds to all preimplantation embryos and also to attached blastocysts (embryo proper and trophoblast). Neuraminidase treatment of blastocyst outgrowths enhances binding of both IIC3 and Ricinus I to the trophoblast; conversely, the binding of wheat germ agglutinin is decreased by this treatment. The results obtained in this study show changes of cell surface carbohydrates during early mouse development and suggest that sialic acid may be masking molecules on the surface of the trophoblast at the time of implantation.