Solubilization and purification of hepatic microsomal trans-2-enoyl-CoA reductase: evidence for the existence of a second long-chain enoyl-CoA reductase

The present study describes the solubilization and purification of a NADPH-specific trans-2-enoyl-CoA reductase from rat liver microsomes. The final preparation was purified to near homogeneity and had a minimal molecular weight of 51,000 +/- 2,000, as judged by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. This enzyme specifically used NADPH, as cofactor, and was chromatographically (2',5'-ADP-agarose) separated from another trans-2-enoyl-CoA reductase which utilized either NADH or NADPH as cofactor. The NADPH-specific trans-2-enoyl-CoA reductase catalyzed the reduction of trans-2-enoyl-CoAs from 4 to 16 carbon units. The Km values for crotonyl-CoA, trans-2-hexenoyl-CoA, and trans-2-hexadecenoyl-CoA were 20, 0.5, and 1.0 microM, while the Km value for NADPH was 10 microM. Although N-ethylmaleimide, heat treatment, and limited proteolysis with trypsin affected the reduction of short-chain (C4) and long-chain (C16) substrates equally, and in spite of the fact that a single protein band was observed on SDS-gels, at the present time one cannot state unequivocally that the purified preparation contained only one reductase. trans-2-Hexenoyl-CoA, for example, did not inhibit the reduction of trans-2-hexadecenoyl-CoA to palmitoyl-CoA and trans-2-decenoyl-CoA to decanoyl-CoA whereas it strongly inhibited the conversion of crotonyl-CoA to butyryl-CoA. The potential implications of this finding are discussed. Finally, the reductase preparation was shown not to contain either heme, nonheme iron, or a flavin prosthetic group.     

Light-induced conformational change in rhodopsin detected by modification of G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation

CNBr treatment of rod outer segments was performed in dark and in light conditions. With the subsequent modified rhodopsin and opsin the cGMP phosphodiesterase activation system was reconstituted. The recombination systems exhibited greatly reduced G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation. The reduction in activity of these three steps of the PDE activation cascade is most significant with modified opsin and is shown to be due to its inability to bind the G alpha subunit. The correlation between the localization of CNBr cleavage in dark and light conditions and these results is strongly indicative that a light-induced conformational change occurs in two extradiscal regions of rhodopsin.     

Linear relationship of phlorizin-binding capacity and hexose uptake during differentiation in a clone of LLC-PK1 cells

With a clone of (Cl 4) of LLC-PK cells, which develop a high capacity for Na+-dependent hexose uptake over time (days) in culture, we show that increasing uptake capacity is paralleled by an increase in the number of phlorizin-binding sites in the population. The linear relationship between binding and hexose transport is the same whether the cells differentiate spontaneously or are induced by either methylisobutylxanthine or hexamethylene bisacetamide. The constancy of the relationship suggests that the primary factor in transport development is the number of transporters in the cells rather than other possible factors like a change in membrane potential or decreased efflux. The Kd for phlorizin binding is .08 +/- .04 microM, and corresponds to Ki of 0.10 microM for transport inhibition. The turnover number of the transporter is estimated to be 170 +/- 40 molecules per second of alpha-methyl glucoside.     

Transepithelial transport of nonelectrolytes in the rabbit mandibular salivary gland

Summary The characteristics of nonelectrolyte secretion by the rabbit mandibular salivary gland have been investigated in anin vitro perfused preparation. The concentrations of¹⁴C-labeled nonelectrolytes were measured in saliva samples collected over a range of flow rates during the secretory response of the gland to continuous acetylcholine infusion. Of the nine nonelectrolytes studied, the two particularly lipid-soluble molecules, ethanol and antipyrine, appeared in the saliva at approximately the same concentration as in the perfusate, regardless of the secretory flow rate. The more polar molecules (urea, ethanediol, thiourea, glycerol, erythritol, mannitol and sucrose) appeared at saliva/perfusate concentration ratios () which showed a strong dependence on flow. With the exception of thiourea, this could be attributed to the combined contributions of diffusion and solvent drag.For the polar nonelectrolytes, estimates have been obtained of both the permeability coefficients of the gland (P) and the solvent-drag filtration coefficients (1–). The relation between 1– and molecular radius suggests that small polar nonelectrolytes and the bulk of the secreted water cross the epithelium via aqueous channels that are approximately 0.8 nm in width. The location of the channels remains uncertain because tissue space measurements indicate that the nonelectrolytes most affected by solvent drag have access to both transcellular and paracellular pathways.     

A benzodiazepine antagonist inhibits the cerebral metabolic and respiratory depressant effects of fentanyl

It is reported that benzodiazepines such as diazepam will stimulate the opiate receptor system and that B-carboline drugs, which are benzodiazepine antagonists, may interact with opiate receptors directly. The ability of 3-hydroxymethyl-B-carboline (3-HMC) to antagonize several parameters of fentanyl anesthesia was tested here in rats. Fentanyl (25 and 100 micrograms/kg iv) produced dose dependent depression of cerebral blood flow (CBF), measured by radioactive microspheres, and cerebral oxygen consumption (CMRO2). These effects were significantly inhibited by 10 mg/kg 3-HMC iv. To test for the specificity of this effect, 3-HMC was also given to rats ventilated with inspire concentrations of 2% halothane. Halothane depressed CMRO2 equally in 3-HMC and vehicle treated rats, indicating no significant effect of the benzodiazepine antagonist. Blood pressure was increased in 3-HMC compared to vehicle treated animals during both fentanyl and halothane anesthesia. CBF was increased in 3-HMC vs vehicle treated rats during halothane anesthesia but this could be accounted for by the elevated blood pressure and lack of cerebral autoregulation rather than a direct cerebrovascular effect. 3-HMC decreased the sleep time and respiratory depressant effects of fentanyl but enhanced the analgesic effects of the opiate, as measured by time to respond to a hot plate stimulus. These results indicate that 3-HMC has the ability to specifically antagonize fentanyl anesthesia. These effects may be produced by an action of 3-HMC at the benzodiazepine receptor and/or by an action of the B-carboline at opioid receptors.     

Interactions between the benzodiazepine receptor antagonist Ro 15-1788 (flumazepil) and the inverse agonist beta-CCE: behavioral studies with squirrel monkeys

The effects of Ro 15-1788 and ethyl-beta-carboline-3-carboxylate (beta-CCE) were studied alone and in combination on the behavioral performances of squirrel monkeys. Under one procedure, performances maintained by food were suppressed by electric shock presentation (punishment or "conflict" procedure). Under a second procedure, responding was maintained either by food or electric shock delivery under a 5-min fixed-interval schedule. Doses of beta-CCE between 0.1 and 3.0 mg/kg, i.m., produced graded decreases in punished responding which were reversed by pretreatment with Ro 15-1788 (1.0 - 10.0 mg/kg, i.m.). Low doses of beta-CCE (0.03 - 0.3 mg/kg, i.m.) increased responding of monkeys maintained by shock presentation, but did not affect food-maintained responding; higher doses of beta-CCE decreased responding under both schedules. These effects of beta-CCE are opposite those produced by the benzodiazepines under this procedure. Ro 15-1788 (1.0 mg/kg i.m.) antagonized the effects of beta-CCE, producing a shift to the right in the dose-response curves. These findings provide further support for the view that beta-CCE and Ro 15-1788 produce effects mediated by the same benzodiazepine receptor recognition site.     

Solvent isotope effects on the reaction catalyzed by yeast hexokinase

The pH dependence of the maximum velocity (V) for the phosphorylation of glucose, the V/Kglucose and the V/KMgATP have been obtained in H2O and 2H2O. In H2O, V decreases below a pK of 5.8, V/Kglucose decreases below a pK of 6.1 and V/KMgATP decreases below a pK of 6.7. In 2H2O, complex behavior is observed for these parameters as a function of pD. The ratios of the parameters in H2O and 2H2O above their respective pK values give solvent deuterium isotope effects of about 1.5-1.7 for all three parameters. When 1,5-anhydromannitol is used as an alternative substrate, an isotope effect different than unity is obtained only for V/K1,5-anhydromannitol which gives a value of about 0.7. Both the complex pH profiles and the relative magnitude of the isotope effects are interpreted in terms of a pH-dependent change in the E X glucose complex.     

Vertebral pathology in the afar australopithecines

Ten vertebral elements from the AL-288 partial hominid skeleton and 11 elements from the AL-333 collection are described. The AL-288 column presents a marked kyphosis at the level of thoracic vertebrae 6 through 10, with pronounced new bone formation on the ventral surfaces of these vertebrae. These features, associated with narrowed disc space and minor osteophytosis, resemble Scheuermann disease in the human. Even though this diagnosis is consistent with a basically human, bipedal locomotor repertoire, the presence of Scheuermann disease suggests that lifting, climbing, or acrobatic activities may have been important in early hominids.