Laminin increases both levels and activity of tyrosine hydroxylase in calf adrenal chromaffin cells

We have investigated the effects of substrate-bound laminin on levels of enzymes of the catecholamine biosynthetic pathway in primary cultures of calf adrenal chromaffin cells. Laminin increases the levels of the enzymes tyrosine hydroxylase, dopamine-beta-hydroxylase, and phenylethanolamine-N-methyl-transferase. This effect is selective, in that levels of other enzymes (lactate dehydrogenase, aromatic amino acid decarboxylase, and acetylcholinesterase) are not increased. The effect of laminin can be blocked by antibodies directed against a fragment of the heparin-binding domain of the molecule, whereas antibodies directed against other fragments do not block the increase in tyrosine hydroxylase. Thus the laminin domain involved in enzyme regulation in chromaffin cells is apparently the same as that previously implicated in laminin's interactions with neurons to potentiate survival and stimulate neurite outgrowth (Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468). The increase in chromaffin cell tyrosine hydroxylase levels is preceded by an activation of the enzyme in which the Vmax (but not the Km) is altered. The effects of laminin appear to be developmentally regulated, since neither activation nor increased levels of tyrosine hydroxylase occur in adult adrenal chromaffin cells exposed to laminin.     

Methyltransferase inhibitors block NGF-regulated survival and protein phosphorylation in sympathetic neurons.

Nerve growth factor (NGF) and elevated K+ concentrations (35 mM) support the survival of the same population of chick embryonic sympathetic neurons. We have used methyltransferase inhibitors, which block protein methylation in intact cells, to investigate the mechanism(s) by which NGF and high K+ exert their effects. Methyltransferase inhibitors selectively blocked NGF-but not high K+-mediated survival of neurons. The ability of neurons, plated on laminin, to respond rapidly to NGF with neurite outgrowth was used to demonstrate that the blockade of the effects of NGF by methyltransferase inhibitors was reversible. At the molecular level, we studied the rapid decrease in phosphorylation of p70, a 70-kd phosphoprotein of sympathetic neurons regulated by both NGF and high K+. Methyltransferase inhibitors blocked the decrease in p70 phosphorylation induced by NGF but not that by high K+. We conclude that the early molecular events of NGF-mediated neuronal survival differ from those of high K+-mediated neuronal survival in that they involve protein methylation, whereas at a later step, possibly at the level of protein phosphorylation, the two pathways leading to survival of sympathetic neurons converge.     

A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase.

Nebularine undergoes hydration at the active site of adenosine deaminase, in a reaction analogous to a partial reaction in the displacement of ammonia from adenosine by water, to generate an inhibitory complex that captures much of the binding affinity expected of an ideal transition-state analogue. Enzyme affinities of several compounds related to nebularine 1,6-hydrate, and to its stable analog 2'-deoxycoformycin, were compared in an effort to identify the structural origins of strong binding. Binding of the stable transition-state analog inhibitor 2'-deoxycoformycin was rendered 9.8 kcal/mol less favorable by removal of substituent ribose, 9.7 kcal/mol less favorable by inversion of the 8-hydroxyl substituent of the diazepine ring, and 10.0 kcal/mol less favorable by removal of atoms 4-6 of the diazepine ring. Binding of the unstable transition-state analog nebularine hydrate was rendered at least 9.9 kcal/mol less favorable by removal of the 6-hydroxyl group and 10.2 kcal/mol less favorable by removal of atoms 1-3 of the pyrimidine ring. In each case, the enzyme exhibited only modest affinity (Kd greater than or equal to 10(-2) M) for the "missing piece", indicating that incorporation of 2 binding determinants within a single molecule permits an additional 7-12 kcal/mol of intrinsic binding energy to be manifested as observed binding energy. These results are consistent with earlier indications that adenosine deaminase may use 10.5 kcal/mol of the intrinsic free energy of binding of the two substrates to place them in positions appropriate for reaction at the active site, overcoming the unfavorable entropy change of -35 eu for the equilibrium of 1,6-hydration of purine ribonucleoside and reducing the equilibrium constant for attainment of the transition state in deamination of adenosine. Thus, adenosine deaminase may achieve up to 8 orders of magnitude of its catalytic power by converting the nonenzymatic, bimolecular, hydration reaction to a monomolecular reaction at its active site. Several new 6-substituted 1,6-dihydropurine ribonucleosides, prepared by photoaddition of formate and by low-temperature addition of organolithium reagents to a derivative of purine ribonucleoside, exhibited Ki values of 9-1400 microM against adenosine deaminase, in accord with the active site's considerable tolerance of bulky leaving groups in substrates. Inhibition by one diastereomer of 6-carboxy-1,6-dihydropurine ribonucleoside was found to be time-dependent, progressing from a weakly bound to a more strongly bound complex.     

NCAM polysialic acid can regulate both cell-cell and cell-substrate interactions

We have proposed previously that the polysialic acid (PSA) moiety of NCAM can influence membrane-membrane apposition, and thereby serve as a selective regulator of a variety of contact-dependent cell interactions. In this study, cell and tissue culture models are used to obtain direct evidence that the presence of PSA on the surface membrane can affect both cell-cell and cell-substrate interactions. Using a neuroblastoma/sensory neuron cell hybrid, it was found that removal of PSA with a specific neuraminidase (endo-N) augments cell-cell aggregation mediated by the L1 cell adhesion molecule as well as cell attachment to a variety of tissue culture substrates. In studies of embryonic spinal cord axon bundling, which involves both cell-cell and cell-substrate interactions, the pronounced defasciculation produced by removal of PSA is most easily explained by an increase in cell-substrate interaction. The fact that in both studies NCAM's intrinsic adhesion function was found not to be an important variable further illustrates that regulation of the cell surface by PSA can extend beyond binding mediated by the NCAM polypeptide.     

Cholinergic differentiation of rat sympathetic neurons in culture: effects of factors applied to distal neurites.

Cholinergic properties are induced in sympathetic neurons by several factors applied to entire neurons in culture. Evidence from work with the rat sweat gland model indicates that factors located in target tissues can induce cholinergic differentiation in vivo. We now report that when leukemia inhibitory factor (LIF), heart cell-conditioned medium (HCCM), or dermal fibroblast-conditioned medium (DFCM) is applied to only distal neurites in compartmented cultures of rat sympathetic neurons, the neurons exhibit an increase in specific choline acetyltransferase activity and a concomitant decrease in levels of tyrosine hydroxylase. LIF, HCCM, and DFCM also induce neurite fasciculation, thus suggesting an additional role of cholinergic switching factors in regulating axon-axon and/or axon-substrate adhesion. These results demonstrate that rat sympathetic neurons have the cellular machinery to respond to cholinergic differentiation cues located in peripheral targets, analogous to the response to nerve growth factor.