Walter Rosenthal

Updated Protocols and Comments on the Purification without Use of Activating Ligands of the Coupling Proteins Ns and Ni of the Hormone Sensitive Adenylyl Cyclase

Ns and Ni have been purified without using NaF and Mg as stabilizing agents (Codina, J., Hildebrandt, J.D., Sekura, R.D., Birnbaumer, M., Bryan, J., Manclark, C.R. and Birnbaumer, L. [1984] J. Biol. Chem. 259, in press). Since the submission of that report, several modifications have been introduced to the purification procedure and additional fractions have been processed from which N proteins are obtained. This article describes the updated protocols and presents methodological details not included in the previous publication. The final products are Ns, the stimulatory N, Ni the inhibitory N, both of subunit structure alpha beta gamma, and a Mr = 40,000 protein of beta gamma composition. They are obtained from human erythrocytes.

[38] Purification of Ns and Ni, the coupling proteins of hormone-sensitive adenylyl cyclases without intervention of activating regulatory ligands

Publisher Summary This chapter presents a detailed description of a procedure for the purification of N s and N i from human erythrocytes and eliminates any possible alteration of the subunit composition of these proteins as might result from the effect of these ligands to induce their subunit dissociation. The chapter explains that it is now recognized that a large number of hormones and neurotransmitters affect their target cells by modulating cyclic adenosine monophosphate (cAMP) formation in either stimulatory or inhibitory fashion. There are two of these couplings or N proteins—an N s (or G s ), mediating the effects of stimulatory hormone–receptor complexes and a N i (or G i ), mediating the effects of inhibitory or attenuating hormone–receptor complexes. The interaction of a hormone–receptor complex with an N protein then results in an increase in the proportion of the N protein in an active vs. inactive conformation or state, and activated N interacts with the catalyst C of adenylyl cyclase eliciting either an increase in catalytic activity (N s ) or an inhibition of activity (N i ). The chapter also explains that structurally, both N s and N i are αβγ heterotrimers. The activation process of both, N s and N i , is dependent on a guanine nucleotide and Mg and seems to involve not only a conformational change but also a subunit dissociation reaction whereby the αβγ heterotrimers dissociate into an activated α* subunit with guanine nucleotide bound to it (α *G ) and αβγ complex.

Chapter 7 Two genes-one disease: The molecular basis of congenital nephrogenic diabetes insipidus

Publisher Summary Congenital nephrogenic diabetes insipidus (NDI) is a rare disorder of the kidney, characterized by the failure to concentrate urine despite normal or elevated levels of the antidiuretic hormone arginine-vasopressin. Identification of the molecular defects underlying congenital NDI is of immediate clinical significance, allowing diagnosis by gene analysis. Gene analysis should be performed in newborns with a family history for NDI and patients of all age groups with a firm diagnosis of congenital NDI, with or without a family history. There is, however, increasing evidence for the assumption that the main defect of many inactivating mutations is the reduced expression of mutant receptors on the cell surface. Here the loss of receptor function occurs regardless of the remaining biological activity of the individual protein. Progress in this field will be crucial for the understanding of the clinical phenotypes of receptor diseases on a molecular level and for the development of therapeutic strategies based on gene transfer.

Molekulare Grundlagen des Diabetes insipidus centralis und renalis

Der Wasserhaushalt aller Primaten und Saugetiere unterliegt einer strengen Kontrolle: Eine Erhohung der Plasmaosmolaritat um <2% oder eine Verminderung des Plasmavolumens um <10% fuhren zu einer vermehrten Freisetzung des antidiuretischen Hormons (ADH). Da es bei Saugetieren zudem eine Gefasverengung hervorruft, wird es auch als Vasopressin (AVP) bezeichnet. Diese Bezeichnung hat sich international durchgesetzt. AVP ist ein zyklisches Nonapeptid (Abb. 9.1), dessen Aminosauresequenz bei fast allen Saugetieren identisch ist. Lediglich beim Schwein findet sich an Position 8 anstelle eines Arginin-ein Lysinrest (Tabelle 9.1). Bei den Reptilien, Amphibien und Wirbellosen wird der Wasserhaushalt ebenfalls uber vasopressinahnliche Peptide reguliert.