The impact of hexose-6-phosphate dehydrogenase (H6PDH) on 11b-hydroxysteroid dehydrogenase (11b-HSD) type 1 activity was investigated upon coexpression in HEK-293 cells. Confocal microscopy analysis indicated colocalisation of both enzymes at the lumenal side of the endoplasmic reticulum (ER) membrane. Functional analysis in intact cells revealed fivefold stimulation of 11b-HSD1 oxoreductase activity and sixfold decrease of dehydrogenase activity upon coexpression with H6PDH, without changing kinetic parameters in cell lysates. Thus, H6PDH directly determines the reaction direction of 11b-HSD1 in intact cells as an oxoreductase without changing intrinsic catalytic properties of 11b-HSD1 by regenerating NADPH in the ER-lumen.

The Monarch (Danaus plexippus) sequesters cardiac glycosides for its chemical defence against predators. Larvae and adults of this butterfly are insensitive towards dietary cardiac glycosides, whereas other Lepidoptera, such as Manduca sexta and Creatonotos transiens are sensitive and intoxicated by ouabain. Ouiabain inhibits the Na+,K+‐ATPase by binding to its α‐subunit. We have amplified and cloned the DNA sequence encoding the respective ouabain binding site. Instead of the amino acid asparagine at position 122 in ouabain‐sensitive insects, the Monarch has a histidine in the putative ouabain binding site, which consists of about 12 amino acids. This change may explain the ouabain insensitivity.

Larvae ofDanaus plexippus feed almost exclusively on milkweed species of the genusAsclepias, whose characteristic secondary metabolites are cardiac glycosides (CGs). Aposematic last-instar larvae were fed with ouabain and other cardiac glycosides of differing polarities. Time course experiments show that ouabain is sequestered in the integument within 48 hr after feeding, whereas midgut tissue and hemolymph function as transient CG storage compartments. About 63% of ouabain was transferred from larvae to the butterflies, whereas 37% of ouabain was lost with larval and pupal exuviae and with the meconium. The main sites of storage in imagines are wings and integument. If mixtures of CGs are fed toD. plexippus larvae, differential sequestration can be observed: The polar ouabain contributes 58.8% of total CGs, followed by digitoxin (19.6%), oleandrin (10.6%), digoxin (4.9%), digoxigenin (4.6%) and proscillaridin A (1.5%). Thus, uptake and sequestration must be selective processes. Uptake of [(3)H]ouabain in vitro by isolated larval midguts was time-, pH-, and temperature-dependent and displayed an activation energy of 49 kJ/mol. Furthermore, the in vitro uptake of ouabain was inhibited (probably competitively) by the structurally similar convallatoxin. These data provide first evidence that ouabain uptake does not proceed by simple diffusion but with the aid of a carrier mechanism, which would explain the differential cardenolide uptake observed in living larvae.

The integrin CD11c/CD18 plays a role in leukocyte and cell matrix adhesion and is highly expressed in certain hematopoietic malignancies. To better characterize ligand binding properties, we panned random peptide phage‐display libraries over purified CD11c/CD18. We identified a phage expressing the circular peptide C‐GRWSGWPADL‐C. C‐GRWSGWPADL‐C phage bound specifically to CD11c/CD18 expressing monocytes but not CD11c/CD18 negative lymphocytes and showed 5 × 103‐fold higher binding to purified CD11c/CD18 than control phage, without binding to CD11b/CD18. Peptide sequence analysis revealed a similar sequence in domain D5 of ICAM‐1 and an alternative, phase‐shifted motif in domain D4. Surface plasmon resonance experiments demonstrated direct interaction of ICAM‐1 and CD11c/CD18. A soluble fusion protein containing the extracellular domain of ICAM‐1 abolished C‐GRWSGWPADL‐C phage binding to CD11c/CD18. Moreover, synthetic monomeric circular peptide C‐GRWSGWPADL‐C bound specifically to CD11c/CD18 and inhibited ICAM‐1 binding. Its rather low binding affinity and inability to displace pentavalent C‐GRWSGWPADL‐C phage from CD11c/CD18 suggests that a multimeric display of the selected peptide is essential for high affinity binding. Using ICAM‐1 deletion constructs, we showed that domain D4 is required for interaction with CD11c/CD18, suggesting that C‐GRWSGWPADL‐C phage binds specifically to CD11c/CD18 by structurally mimicking the interaction site on D4 of ICAM‐1.

By interconverting glucocorticoids, 11␤-hydroxysteroid dehydrogenase type 1 (11␤-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11␤-HSD1 plays a potential role in 7-ketocholesterol metabolism. Here we investigated the role of the Nterminal region on enzymatic activity and addressed the relevance of 11␤-HSD1 orientation into the endoplasmic reticulum (ER) lumen. Previous studies revealed that the luminal orientation of 11␤-HSD1 and 50-kDa esterase/arylacetamide deacetylase (E3) is determined by their highly similar N-terminal transmembrane domains. on enzymatic activity, suggesting that these residues are responsible for the observed stabilizing effect of the N-terminal membrane anchor on the catalytic domain of 11␤-HSD1. Moreover, activity measurements in intact cells expressing wildtype 11␤-HSD1, facing the ER lumen, or mutant K5S/ K6S, facing the cytoplasm, revealed that the luminal orientation is essential for efficient oxidation of cortisol. Furthermore, we demonstrate that 11␤-HSD1, but not mutant K5S/K6S with cytoplasmic orientation, catalyzes the oxoreduction of 7-ketocholesterol. 11␤-HSD1 and E3 constructs with cytosolic orientation of their catalytic moiety should prove useful in future studies addressing the physiological function of these proteins.In humans, 11␤-HSD1 1 catalyzes the reduction of biologically inactive cortisone to active cortisol, thereby playing an essential role in the local activation of the glucocorticoid receptor. Recent animal experiments provided insight into the pathophysiological role of 11␤-HSD1. Mice deficient of 11␤-HSD1 were resistant to hyperglycemia induced by obesity or stress (1), whereas transgenic mice overexpressing 11␤-HSD1 developed visceral obesity with insulin resistance and dyslipidemia. In addition, overexpression of 11␤-HSD1 in adipose tissue caused salt-sensitive hypertension mediated by an activated renin-angiotensin system (2, 3). Experiments in obese and diabetic mice treated with a specific 11␤-HSD1 inhibitor showed reduced blood glucose levels and increased insulin sensitivity (4, 5). Therefore, 11␤-HSD1 is currently considered a promising drug target for the treatment of cognitive dysfunction in elderly men and patients with obesity and type 2 diabetes mellitus (6, 7). However, whether 11␤-HSD1 is indeed a suitable target for therapeutic treatment of excessive glucocorticoid actions remains to be tested.Recently, we provided evidence that 11␤-HSD1 plays a role in the rapid hepatic metabolism of 7-ketocholesterol (7KC) (8), the major oxysterol in processed cholesterol-rich food and, after 27-hydroxycholesterol, in advanced atherosclerotic plaques (9 -11). In rats treated with 7KC and the 11␤-HSD inhibitor carbenoxolone, 7KC tended to accumulate in liver and plasma (8). In addition, 11␤-HSD1 seems to catalyze the interconversion of 7-hydroxylated dehydroepiandrosterone metabolites (12) and has a potential role in biotransformation by reducing reactive ketones such as the potent tob...