In vitro acylation of rat gastric mucus glycoprotein with [3H]palmitic acid

The incorporation of fatty acids into gastric mucus glycoproteins was studied by incubating rat gastric mucosal cell suspensions with [9,10-3H]palmitic acid and [3H]proline. The mucus glycoprotein polymer, secreted into the growth medium (extracellular) and that contained within the cells (intracellular), was purified from the other components of the secretion, thoroughly delipidated, and then analyzed for the radiolabeled tracers. Both pools of mucus glycoprotein, incubated in the presence of [3H]palmitic acid, contained radioactive label which could not be removed by gel filtration, CsCl density gradient centrifugation, sodium dodecyl sulfate-gel electrophoresis, or lipid extraction. Treatment of the purified mucus glycoprotein with 1 M hydroxylamine or 0.3 M methanolic KOH released the radioactivity, thus indicating that [3H]palmitic acid was covalently bound by ester linkage to the glycoprotein. The released radioactivity was associated mainly (87%) with palmitic acid. The incorporation ratio of [3H]proline to [3H]palmitic acid was 0.12:1.0 in the extracellular glycoprotein and 1.38:1.0 in the intracellular glycoprotein, which suggested that acylation of mucus glycoprotein occurs in the intracellular compartment after completion of its polypeptide core. The fact that incorporation of [3H]palmitic acid was greater in the glycoprotein subunits than in the glycoprotein polymer indicates that acylation takes place near the end of subunit processing but before their assembly into the high molecular weight mucus glycoprotein polymer.     

Acylation of bovine rhodopsin by [3H]palmitic acid

Bovine retinas or preparations of rod outer segments incorporate [3H]palmitic acid into rhodopsin. The incorporation is both time- and temperature-dependent. The major product retains the chromatographic and electrophoretic properties of rhodopsin and remains photosensitive as demonstrated by alteration of its chromatographic behavior upon exposure to light. The incorporated radioactivity resists extraction with organic solvents and is not dissociated from the protein by detergents or under the denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive free fatty acid can, however, be released by alkaline hydrolysis. Hydroxylamine treatment yields a mixture of the free fatty acid and the fatty acyl hydroxamate. These results demonstrate the formation of an ester bond between [3H]palmitic acid and rhodopsin. Cycloheximide fails to inhibit the incorporation. This finding along with the ability of rod outer segments to support the incorporation point to the acylation of rhodopsin as a late post-translational event.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

Metabolism of [3H]leupeptin by rat liver

Tritium-labeled leupeptin was used to study how this tripeptide proteinase inhibitor interacts with the liver, including the mechanism of its transport into the cell, its subcellular distribution after uptake, and its metabolism once in the tissue. Experiments were done in situ and in a perfused liver. At low concentrations (1 to 10 μm) the uptake of radioactive inhibitor was competed by chemically reduced leupeptin. At high concentrations at least up to 400 μm the uptake was directly proportional to the external concentration of tripeptide. Entry into the tissue essentially stopped at low temperature (<21 °C). [³H]Leupeptin initially was located in the soluble fraction of the liver homogenate and by 15 to 30 min became concentrated in the lysosome-rich fraction. During 2 h of perfusion almost 50% of [³H]leupeptin that had entered the liver was secreted intact into the bile. In addition, a portion of the leupeptin that remained in the liver was degraded during this time period.     

Multiple opioid receptors: An examination of the dissociation of [3H]leucine enkephalin from rat brain membranes

The experiments reported in this paper address the hypothesis that [3H]leucine enkephalin labels both mu and delta receptors. As reported by other workers, this peptide dissociates from rat brain membranes in a biphasic manner. This is consistent with a two site binding model which hypothesizes that the peptide labels both opioid mu and delta receptors from which it dissociates at different rates. To test this hypothesis, we determined the dissociation of bound ligand from rat brain membranes incubated to equilibrium with [3H]leucine enkephalin in the absence and presence of 100 nM morphine. The data were not significantly different. We conclude that the biphasic off-kinetics of [3H]leucine enkephalin is not evidence for a two-site binding model.     

H2 histaminic receptors in rat cerebral cortex. 3. Inhibition of [3H]histamine by H2 agonists

The binding of [3H]histamine to H2 receptors in homogenates of rat cerebral cortex is inhibited by 11 H2 agonists in a characteristic and unique manner. At low concentrations of the radioligand (less than 1.5 nM), the inhibitory profiles of individual agonists (A) are distinctly biphasic; specific binding is well described in most cases by the empirical expression Y = F1K1/(K1 + [A]) + F2K2/(K2 + [A]), in which F1 and F2 sum to 1. Maximal inhibition is the same for all agonists. Since values of F2 vary from 0.42 to 0.90, the agonist appears to determine the equilibrium distribution of receptors between two states of affinity. Ratios of apparent affinity (K2/K1) vary from 204 to 3 090 000, and there is no correlation between values of K1 and K2. Compounds lacking H2 activity, including structural analogues of histamine and dimaprit, reveal a Hill coefficient of 1 and inhibit the radioligand only weakly. For six agonists, values of K2 agree and correlate well (P = 0.00047) with H2 pharmacological potency (EC50) in the guinea pig right atrium; for the others, K2 is less than EC50 by 15-61-fold. Four observations suggest that the inhibition corresponding to F1 is allosteric and cooperative: the dissociation constant of the radioligand appears to vary in the presence of an unlabeled agonist, absolute levels of binding corresponding to F1, as defined by dimaprit, decrease at higher concentrations of [3H]histamine, F1 for dimaprit is reduced from 0.48 to 0.32 by 2-methylhistamine (F1 = 0.27) at a concentration of 20 nM (approximately K1(0.5) K2(0.5) for 2-methylhistamine), but the increase in K1 for dimprit is at least 100-fold less than expected from competitive effects, and 1 equiv of some agonists appears to preclude access of [3H]histamine to more than 1 equiv of receptors, with no evidence that an appreciable fraction of the unlabeled drug is bound. Noncompetitive effects also may account in part for the inhibition corresponding to F2.     

H2 histaminic receptors in rat cerebral cortex. 2. Inhibition of [3H]histamine by H2 antagonists

Sites labeled by [3H]histamine in homogenates of rat cerebral cortex reveal a pharmacological specificity typical of H2 receptors. Fourteen H2 antagonists inhibit the specific binding of the radioligand to the same level; Hill coefficients are near or equal to one for five compounds and markedly lower for nine. The binding patterns of individual antagonists (A) are well described by the empirical expression Y = F1K1/(K1 + [A]) + F2K2/(K2 + [A]), in which F1 and F2 sum to 1; F2 is 0 for those drugs that reveal a Hill coefficient of 1. Concentrations of A that reduce specific binding by 50% (IC50) correlate well (r = 0.991; P less than 0.00001) and show good numerical agreement with potencies reported for inhibition of the response to histamine in H2-mediated systems. The correlation is poorer when IC50 is replaced by either K1 (r = 0.973) or K2 (r = 0.921) for those antagonists that reveal both; the antihistaminic activity of the drug thus appears not to be associated preferentially with one or other class of sites. Since F2 varies from 0.16 to 0.60 among those antagonists that discern heterogeneity, the antagonist appears to determine the distribution of sites between the two classes. Moreover, a correlation among antagonists between values of K1 and K2 (r = 0.975; P = 0.00001) suggests that the apparent heterogeneity reflects different conformers within an otherwise homogeneous population. H2 antagonists appear to be noncompetitive with respect to each other and to the radioligand: one antagonist has relatively little effect on the values of K1, K2, and F2 revealed by another; also, estimates of K1 and K2 are independent of the concentration of [3H]histamine between 1.3 and 10 nM, although the radioligand exhibits an apparent dissociation constant of 3.9 nM [Steinberg, G. H., Eppel, J. G., Kandel, M., Kandel, S. I., & Wells, J. W. (1985) Biochemistry (preceding paper in this issue)].     

Conversion of 19-oxo[2 beta-2H]androgens into oestrogens by human placental aromatase. An unexpected stereochemical outcome.

Aromatase is a cytochrome P-450 enzyme that catalyzes the conversion of androgens into oestrogens via sequential oxidations at the 19-methyl group. Despite intensive investigation, the mechanism of the third step, conversion of the 19-aldehydes into oestrogens, has remained unsolved. We have previously found that a pre-enolized 19-al derivative undergoes smooth aromatization in non-enzymic model studies, but the role of enolization by the enzyme in transformations of 19-oxoandrogens has not been previously investigated. The compounds 19-oxo[2 beta-2H]testosterone and 19-oxo[2 beta-2H]androstenedione have now been synthesized. Exposure of either of these compounds to microsomal aromatase, in the absence of NADPH, for an extended period led to no significant 2H loss or epimerization at C-2, leaving open the importance of an active-site base. However, in the presence of NADPH there was an unexpected substrate-dependent difference in the stereoselectivity of H loss at C-2 in the enzyme-induced aromatization of 19-oxo[2 beta-2H]-testosterone versus 19-oxo[2 beta-2H]androstenedione. The aromatization results for 17 beta-ol derivative 19-oxo[2 beta-2H]-testosterone correspond to about 1.2:1 2 beta-H/2 alpha-H loss from unlabelled 19-oxotestosterone. In contrast, aromatization results for 19-oxo[2 beta-2H]androstenedione correspond to at least 11:1 2 beta-H/2 alpha-H loss from unlabelled 19-oxoandrostenedione. This substrate-dependent stereoselectivity implies a direct role for an enzyme active-site base in 2-H removal. Furthermore, these results argue against the proposal that 2 beta-hydroxylation is the obligatory third step in aromatase action.     

Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine

The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after [3H]uridine injection or by the de novo pathway as shown after [3H]orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium--that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either [5-3H]uridine or [5-3H]orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloracetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after [3H]uridine and 72% after [3H]orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A+ RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after [3H] uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant. After [3H]-orotic acid injection, the number of silver grains over the nucleolus was negligible at all levels, whereas over the nucleoplasm the number was low in crypt cells, but high in villus cells with a peak in mid villus. The interpretation is that, except for a small amount of label incorporated into DNA from either precursor by crypt cells, the bulk of the label is incorporated into RNA as follows. In the crypts, cells make almost exclusive use of uridine, that is, of the salvage pathway, for the synthesis of ribosomal RNA in the nucleolus and of messenger and transfer RNA in the nucleoplasm. However, when cells pass from crypt to villus, they mainly utilize orotic acid--i.e., the de novo pathway--for the synthesis of messenger and transfer RNA within the nucleoplasm.     

Effect of arachidonic acid on [3H]d-aspartate outflow in the rat hippocampus

The aim of this study was to investigate the effect of arachidonic acid on [³H]d-aspartate outflow in rat hippocampus synaptosomes and slices. Arachidonic acid 1) increased basal outflow of [³H]d-aspartate in both synaptosomes and slices, and 2) increased K⁺-evoked overflow in slices but not in synaptosomes. The latter effect was dependent (at least in part) on arachidonic acid metabolism, most likely mediated by lipo-oxygenase metabolites and free radical production. It was prevented by nordihydroguaiaretic acid but not by indomethacin, and was significantly reduced by free radical scavengers (superoxide-desmutase and catalase). This effect was dependent upon stimulation since it could not be observed after a continuous perfusion of arachidonic acid in the absence of stimulation. Furthermore, it was long-lasting since a 30 min perfusion of arachidonic acid was sufficient to exert a significant effect on a stimulation following termination of the application.     

Properties of [3H] diazepam binding to rat peritoneal mast cells

Benzodiazepine binding to rat peritoneal mast cells was investigated using [³H] diazepam as the radioactive probe. The specific binding of [³H] diazepam reaches equilibrium within 10–15 min, is saturable and is linear with cell number. Scatchard analysis of equilibrium binding indicates the existence of only one class of binding sites with a K_D = 90 ± 10 nM and B_max of 261 ± 60 fmoles/10⁶ cells. The binding of [³H] diazepam is temperature dependent, the highest amount is bound at 0°C and shows a pH-optimum between pH 6.8 – 7.4. The binding of [³H] diazepam is reversible with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2 ± 0.2}\text{min.}$ Based on the relative potency of clonazepam and Ro5-4864 in displacing the specific [³H] diazepam binding, the binding sites in the mast cell are similar to those in the peripheral tissues like lung, liver, and kidney and are different from those in the brain. These data indicate that the mast cells have benzodiazepine binding sites which are of the peripheral type.     

Metabolism of 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose in yeast and chick-embryo cells.

2-Deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose have been prepared by tritiation of the corresponding unlabeled 2-fluoro sugars. The tritiated 2-fluoro sugars are phosphorylated and activated by UTP and by GTP to yield UDP-2-deoxy-2-fluoro-D-[3H]glucose, UDP-2-deoxy-2-fluoro-D-[3H]mannose, GDP-2-deoxy-2-fluoro-D-[3H]glucose and GDP-2-deoxy-2-fluoro-D-[3H]mannose in both cell types. The nucleotide derivatives could also be labeled in the nucleotide moiety by feeding the cells with [14C]uridine or [14C]guanosine in the presence of unlabeled 2-fluoro sugar. No evidence was obtained for metabolic steps in which the six-carbon chain of 2-fluoro sugars was not preserved. No epimerisation of the label to 2-deoxy-2-fluoro-D-[3H]galactose could be observed by radioactive gas-liquid chromatography of the enzymatic cleavage products of the different 2-fluoro sugar metabolites isolated from either cell type. Yeast and chick embryo cells both incorporate 2-deoxy-2-fluoro-D-[3H]glucose and 2-deoxy-2-fluoro-D-[3H]mannose specifically into glycoproteins, although this incorporation is very low when compared to the incorporation of 2-deoxy-D-[3H]glucose.     

Enkephalin convertase: characterization and localization using [3H]guanidinoethylmercaptosuccinic acid

Enkephalin convertase (carboxypeptidase E,H; EC 3.4.17.10) is a carboxypeptidase B-like enzyme which appears to be physiologically associated with the biosynthesis of the enkephalins and certain other peptides. We have localized enkephalin convertase in the brain and other tissues autoradiographically by labeling studies with [3H]guanidinoethylmercaptosuccinic acid ([3H]GEMSA). In the brain, [3H]GEMSA localizations parallel enkephalin distribution but with certain exceptions, suggesting a role in relation to other peptides. In the pancreas, [3H]GEMSA binding sites are localized to the islets suggesting an involvement in insulin, glucagon, or somatostatin formation. The selective concentration of [3H]GEMSA grains in cardiac atria suggests a link to atrial natriuretic factor.