Affinity labeling of bovine liver glutamate dehydrogenase with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate

Bovine liver glutamate dehydrogenase reacts with 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TA-5'-DP) and 5'-triphosphate (8-BDB-TA-5'-TP) to yield enzyme with about 1 mol of reagent incorporated/mol of enzyme subunit. The modified enzyme is catalytically active but has decreased sensitivity to inhibition by GTP, reduced extent of activation by ADP, and diminished inhibition by high concentrations of NADH. Since modified enzyme, like native glutamate dehydrogenase, reversibly binds more than 1 mol each of ADP and GTP, it is unlikely that 8-BDB-TA-5'-TP reacts directly within either the ADP or GTP regulatory sites. The rate constant for reaction of enzyme exhibits a nonlinear dependence on reagent concentration with KD = 89 microM for 8-BDB-TA-5'-TP and 240 microM for 8-BDB-TA-5'-DP. The ligands ADP and GTP alone and NADH alone produce only small decreases in the rate constant for the reaction of enzyme with 8-BDB-TA-5'-TP, but the combined addition of 5 mM NADH + 200 microM GTP reduces the reaction rate constant more than 10-fold and the reagent incorporation to about 0.1 mol/mol of enzyme subunit. These results suggest that 8-BDB-TA-5'-TP reacts as a nucleotide affinity label in the region of the GTP-dependent NADH regulatory site of bovine liver glutamate dehydrogenase.     

Purification and properties of eIF-2 phosphatase

Eukaryotic initiation factor 2 (eIF-2) phosphatase has been purified 840-fold to apparent homogeneity from rabbit reticulocyte lysate. Native eIF-2 phosphatase has a Mr = 98,000, pI = 6.1, s20,w = 5.1, and a Stokes radius = 38 A. A subunit composition of one 60,000-dalton polypeptide and one 38,000-dalton polypeptide is indicated. The Km for [32P]eIF-2 is 30 microM and the Vmax = 1.1 nmol of phosphate released/min/microgram. The 38,000-dalton subunit of eIF-2 phosphatase does not co-electrophorese with the catalytic subunit of liver phosphorylase phosphatase, a type 1 protein phosphatase. The specificity of eIF-2 phosphatase for phosphorylation sites on th alpha- and beta-subunits of eIF-2 appears to be determined by the environment of the phosphatase and substrate. Both the alpha- and beta-subunits of [32P]eIF-2 are rapidly dephosphorylated by the purified phosphatase. In unfractionated lysate and in unfractionated lysate supplemented with an equivalent activity of the purified phosphatase, only the alpha-subunit of eIF-2 is dephosphorylated. This indicates other factors are present in the lysate which govern the dephosphorylation of eIF-2.     

Eukaryotic initiation complex formation. Evidence for two distinct pathways.

Two distinct pathways have been elucidated which lead to the formation of an AUG-dependent initiation complex. One pathway involves the use of initiation factor M1 (IF-M1) to promote AUG-dependent binding of the initiator tRNA to the 40 S subunit, followed by joining of the 60 S subunit in the presence of IF-M2A, IF-M2B, and GTP. The second pathway involves the IF-MP-directed binding of initiator tRNA to the 40 S subunit via a ternary complex of IF-MP-GTP-Met-tRNAf. This reaction does not require AUG codon. However, subsequent formation of an 80 S initiation complex (as determined by methionyl-puromycin synthesis) required AUG as well as IF-M2A, IF-M2B, and GTP. Since both pathways require the same complementary initiation factors (at the same level), it would appear that the only difference is the manner in which the initiator tRNA is bound to the 40 S subunit, either by IF-M1 or IF-MP. Examination of the requirements for endogenous mRNA-directed methionyl-puromycin synthesis indicates a greater difference between IF-MP and IF-M1 in that only IF-MP was capable of forming an 80 S initiation complex which was sensitive to puromycin.     

MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments

MAP2 and tau exhibit microtubule-stabilizing activities that are implicated in the development and maintenance of neuronal axons and dendrites. The proteins share a homologous COOH-terminal domain, composed of three or four microtubule binding repeats separated by inter-repeats (IRs). To investigate how MAP2 and tau stabilize microtubules, we calculated 3D maps of microtubules fully decorated with MAP2c or tau using cryo-EM and helical image analysis. Comparing these maps with an undecorated microtubule map revealed additional densities along protofilament ridges on the microtubule exterior, indicating that MAP2c and tau form an ordered structure when they bind microtubules. Localization of undecagold attached to the second IR of MAP2c showed that IRs also lie along the ridges, not between protofilaments. The densities attributable to the microtubule-associated proteins lie in close proximity to helices 11 and 12 and the COOH terminus of tubulin. Our data further suggest that the evolutionarily maintained differences observed in the repeat domain may be important for the specific targeting of different repeats to either alpha or beta tubulin. These results provide strong evidence suggesting that MAP2c and tau stabilize microtubules by binding along individual protofilaments, possibly by bridging the tubulin interfaces.     

E1B 19,000-Molecular-Weight Protein Interacts with and Inhibits CED-4-Dependent, FLICE-Mediated Apoptosis

Genetic studies of the nematode Caenorhabditis elegans (C. elegans) have identified several important components of the cell death pathway, most notably CED-3, CED-4, and CED-9. CED-4 directly interacts with the Bcl-2 homologue CED-9 (or the mammalian Bcl-2 family member Bcl-x_L) and the caspase CED-3 (or the mammalian caspases ICE and FLICE). This trimolecular complex of CED-4, CED-3, and CED-9 is functional in that CED-9 inhibits CED-4 from activating CED-3 and thereby inhibits apoptosis in heterologous systems. The E1B 19,000-molecular weight protein (E1B 19K) is a potent apoptosis inhibitor and the adenovirus homologue of Bcl-2-related apoptosis inhibitors. Since E1B 19K and Bcl-x_L have functional similarity, we determined if E1B 19K interacts with CED-4 and regulates CED-4-dependent caspase activation. Binding analysis indicated that E1B 19K interacts with CED-4 in a Saccharomyces cerevisiae two-hybrid assay, in vitro, and in mammalian cell lysates. The subcellular localization pattern of CED-4 was dramatically changed by E1B 19K, supporting the theory of a functional interaction between CED-4 and E1B 19K. Whereas expression of CED-4 alone could not induce cell death, coexpression of CED-4 and FLICE augmented cell death induction by FLICE, which was blocked by expression of E1B 19K. Even though E1B 19K did not prevent FLICE-induced apoptosis, it did inhibit CED-4-dependent, FLICE-mediated apoptosis, which suggested that CED-4 was required for E1B 19K to block FLICE activation. Thus, E1B 19K functions through interacting with CED-4, and presumably a mammalian homologue of CED-4, to inhibit caspase activation and apoptosis.     

Teaching glucocorticoid negative feedback and adrenocortical regulation using a classic paper by Dr. Dwight Ingle

The American Physiological Society (APS) Legacy Project and its accompanying Essays on APS Classic Papers have allowed the scientific community on-line access to the entire collection of APS publications since their inception in 1898 (http://www.the-aps.org/publications/legacy/ and http://www.the-aps.org/publications/classics/). The availability of the classic physiological studies provides a unique teaching opportunity. The classic paper of Dr. Dwight Ingle represents just such a study. Dr. Ingle demonstrated that, using only purified extracts of the pituitary (ACTH) and adrenal cortex (corticosterone) and hypophysectomized rats, he could establish several of the basic principles of the control of adrenal function and glucocorticoid negative feedback that are now standard teaching material in endocrinology. An annotated figure from Dr. Ingle's paper is provided, which, when assigned to undergraduate or graduate students, will allow discovery learning. Furthermore, the brilliance and imagination of the physiologists of the last century are highlighted, which allows an appreciation of the seminal work of our predecessors.     

Steroidogenesis in human aldosterone-secreting adenomas and adrenal hyperplasias: effects of hypoxia in vitro

The synthesis of adrenal steroids requires molecular oxygen. Because arterial hypoxemia is a common clinical condition, the purpose of the present study was to examine steroidogenesis in vitro under physiological changes in O(2) tension (Po(2)) in cells from human adrenal glands with aldosterone-secreting adenomas (ASA; n=3) or with bilateral adrenal hyperplasia causing Cushing's syndrome (n=4). A decrease in Po(2) from 150 mmHg (mild hyperoxia) to 80 mmHg had minimal effect on steroid production. A reduction to 40 mmHg (still well within the physiological range) significantly inhibited cAMP- and ACTH-stimulated aldosterone, cortisol, and dehydroepiandrosterone (DHEA) production from ASA. Furthermore, cortisol and DHEA production in cells from histologically normal tissue, adjacent to ASA and from bilateral adrenal hyperplasias, was also inhibited under a Po(2) of 40 mmHg. We conclude that physiological decreases in Po(2) to levels typical for adrenal venous Po(2) under mild hypoxia inhibit steroidogenesis. These studies may have implications for oxygen therapy in critically ill patients with functional adrenal insufficiency, as well as for therapeutic options in patients with adrenal neoplasms.     

Ultrastructure of the nephron of the one-humped camel, Camelus dromedarius

The nephron of the one-humped camel Camelus dromedarius was investigated by light and transmission electron microscopy. Besides the many features common to other mammalian kidneys, the nephron of the camel is unique in having an unusually thick basal lamina underlying the epithelial cells of the nephron, the thickest being found in part of the parietal layer of Bowman's capsule and the thin limb of the loop of Henle. In the latter, the membrane usually appears lamellated and contains numerous tiny vesicles. In other parts of the nephron, the basal lamina usually has a homogenous appearance. The possible significance of the thickening of the basal lamina is discussed in relation to the general high renal efficiency of the camel.