No abstract

Treatment of turkey erthrocyte membranes with cholera toxin caused an enhancement of the basal and catecholamine-siimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activities. Both of these activities required the presence of GTP. The toxin effect on the adenylate cyclase activity coincided with an inhibition of the catecholamine-stimulated guanosinetriphosphatase activity. Inhibition of the guanosinetriphosphatase, as well as enhancement of the adenylate cyclase activity, showed the same dependence on cholera toxin concentrations, and the effect of the toxin on both activities was dependent on the presence of NAD.It is propsed that continuous GTP hydrolysis at the regulatory guanyl nucleotide site,is an essential turn-off mechanism, The results have shown that the toxin concurrently inhibits the GTPase activity and enhances the GTP-dependent adenylate cyclase activity. Furthermore, the effects of GTP on the adenylate cyclase in preparations treated with cholera toxin resembled those seen with the hydrolysis-resistant analog Gpp(NH)p in untreated preparations. We therefore propose that cholera toxin activates the adenylate cyclase via an inhibition of the turn-off GTPase reaction. ['y-32P]GTP was prepared by the method of Glynn and Chappell (14). MATERIALS AND METHODSTurkey Erythrocyte Membranes. These were prepared by digestion of the nuclei with DNase, as described for frog erythrocytes (15), with the following modifications: the pH of the Tris buffer used for lysis and for washes of the membranes was 7.9 at 230 and 2-mercaptoethanol (2 mM) was used instead of dithiothreitol. The membranes were stored in liquid nitrogen and thawed on the day of the experiment.Toxin Treatment. Adenylate cyclase in membrane preparations from turkey erthrocytes was activated with cholera toxin (120 ,g/ml) that had been incubated for 15 min at 370 in turkey erythrocyte cytosol containing 0.6 mM dithiothreitol.t The cytosol was prepared from packed erythrocytes lysed by freezing in liquid nitrogen, followed by thawing at 370, susAbbreviations: Gpp(NH)p, guanosine 5'-(f,-y-imino)triphosphate; App(NH)p, adenosine 5'-(,By-imino)triphosphate; GTPase, guanosinetriphosphatase; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. * To whom correspondence should be addressed.

No abstract

This article describes a new concept of medium- and long-range cyclization of peptides through "backbone cyclization." In this approach, conformational constraints are conferred on a peptide by linking omega-substituted alkylidene chains replacing N(alpha) or C(alpha) hydrogens in a peptidic backbone. Backbone cyclization, which is divided into N-backbone and C-backbone cyclizations, allow for new modes of cyclization in addition to the classical ones that are limited to cyclization through the side chains and/or the amino or carboxyl terminal groups. The article also describes the application of the N-backbone cyclization to the active region of substance P. Conformational constraints of this peptide by the classical cyclization modes led to inactive analogues whereas N-backbone cyclization provided an active, selective, and metabolically stable analogue.

The existence of a third tachykinin receptor (SP‐N) in the mammalian nervous system was demonstrated by development of highly selective agonists. Systematic N‐methylation of individual peptide bonds in the C‐terminal hexapeptide of substance P gave rise to agonists which specifically act on different receptor subtypes. The most selective analog of this series, succinyl‐[Asp6,Me‐Phe8]SP6‐11, elicits half‐maximal contraction of the guinea pig ileum through the neuronal SP‐N receptor at a concentration of 0.5 nM. At least 60,000‐fold higher concentrations of this peptide are required to stimulate the other two tachykinin receptors (SP‐P and SP‐E). The action of selective SP‐N agonists in the guinea pig ileum is antagonized by opioid peptides, suggesting a functional counteraction between opiate and SP‐N receptors. These results indicate that the tachykinin receptors are distinct entities which may mediate different physiological functions.

Two flat revertants have been isolated from mutagen-treated populations of Kirsten murine sarcoma virus (KiMuSV)-transformed NIH/3T3 cells. These revertants, which appear to be cellular variants resistant to transformation by the KiMuSV oncogene v-Ki-ras, contain Ki-MuSV-specific DNA, elevated levels of the v-Ki-ras gene product p2l, and rescuable transforming virus. Cell hybridization studies indicated that the revertant phenotype is dominant in hybrids between revertant cells and cells transformed by Ki-MuSV or the closely related Harvey MuSV and BALB MuSV. Analysis of hybrid cells resulting from the fusion of these revertants to cell lines transformed by other retroviruses showed that the action of certain oncogenes structurally unrelated to v-Ki-ras also could be suppressed. Thus, there appear to be functional relationships and diversities among transforming genes (oncogenes) not readily apparent from their structural characteristics.Recent molecular studies have defined the structure of a number of transforming genes (oncogenes) and their products in considerable detail (reviewed in ref. 1). All of the known retroviral oncogenes are closely related to sequences present in normal vertebrate cells (cellular proto-oncogenes), from which they appear to have arisen (1, 2). The viral oncogenes associated with Kirsten and Harvey murine sarcoma viruses (Ki-and Ha-MuSVs), v-Ki-ras and v-Ha-ras, for example, are known to encode similar phosphorylated 21,000-dalton proteins (designated p2ls) with guanine nucleotide-binding activities (3-6). v-Ki-ras and v-Haras are structurally related to highly conserved cellular sequences (proto-oncogenes), some of which may be associated with the etiology of certain human tumors (refs. 7 and 8; reviewed in refs. 9 and 10).In spite of the relatively large body of information concerning the molecular structure of retrovirus oncogenes and the proteins that they specify, comparatively little is known about the cellular components with which these proteins interact and the mechanism(s) by which they transform cells. The nature of the cellular components involved in the expression of transformation can be defined theoretically by the isolation and molecular characterization of flat nontransformed variants (revertants) from populations of retrovirus-transformed cells. Although a number of such flat revertants have been isolated from cells transformed by retroviruses (refs. 11-21; reviewed in ref. 22), the great majority of these has been shown to lack expression of functioning viral oncogenes. A few revertant cell lines containing apparent alterations in host cell genomes have been isolated (13, 21); however, little is known about the specific cellular factors involved in the reversion process.This report describes two cellular revertants that are resistant to transformation by Ki-MuSV and certain other retroviruses. These revertants may be used to study the mechanism of transformation by the v-Ki-ras oncogene and to reveal functional relationships among different viral oncogene...

ABSTRACTformed is inositol trisphosphate, and that this product is rapidly hydrolyzed by a specific phosphomonoesterase. Introduction of inositol trisphosphate to the intact photoreceptor cell mimics the effect of light, and bisphosphoglycerate, which inhibits inositol trisphosphate hydrolysis, enhances the effects of inositol trisphosphate and of dim light. The interaction of photoexcited rhodopsin with a G protein is thus similar in both vertebrate and invertebrate photoreceptors. These G proteins, however, activate different photoreceptor enzymes: phospholipase C in invertebrates and cGMP phosphodiesterase in vertebrates.

Excitation of fly photoreceptor cells is initiated by photoisomerization of rhodopsin to the active form of metarhodopsin. Fly metarhodopsin is thermostable, does not bleach, and does not regenerate spontaneously to rhodopsin. For this reason, the activity of metarhodopsin must be stopped by an effective termination reaction. On the other hand, there is also a need to restore the inactivated photopigment to an excitable state in order to keep a sufficient number of photopigment molecules available for excitation. The following findings reveal how these demands are met. The photopigment undergoes rapid phosphorylation upon photoconversion of rhodopsin to metarhodopsin and an efficient Ca2+ dependent dephosphorylation upon regeneration of metarhodopsn to rhodopsin. Phosphorylation decreases the ability of metarhodopsin to activate the guanine nucleotide-binding protein.Binding of 49-kDa arrestin further quenches the activity of metarhodopsin and protects it from dephosphorylation. Lightdependent binding and release of 49-kDa arrestin from metarhodopsin-and rhodopsin-containing membranes, respectively, directs the dephosphorylation reaction toward rhodopsin. This ensures the return of phosphorylated metarhodopsin to the rhodopsin pool without initiating transduction in the dark. Assays of rhodopsin dephosphorylation in the Drosophila retinal degeneration C (rdgC) mutant, a mutant in a gene previously cloned and predicted to encode a serine/threonine protein phosphatase, reveal that phosphorylated rhodopsin is a major substrate for the rdgC phosphatase. We propose that mutations resulting in either a decrease or an improper regulation of rhodopsin phosphatase activity bring about degeneration of the fly photoreceptor cells. While transduction in fly photoreceptors bears many similarities to transduction in vertebrate rods, it also manifests several important differences. In particular, the target for the rhodopsin-activated G protein is a phospholipase C (8-10); a second messenger appears to be inositol trisphosphate (11,12) and there are at least two types of light-dependent ion channels, one of which is highly permeable to Ca2+ (13).Invertebrate photoreceptor channels open (14), whereas vertebrate rod channels close, in response to illumination. Thus, light increases the free Ca2+ in invertebrate photoreceptors and decreases Ca2+ concentration in vertebrate rods. Experiments on intact Drosophila eyes identified light-dependent phosphorylation of three eye-specific proteins of 39, 49, and 80 kDa (15).In the blowfly Calliphora, illumination of retinal homogenate induced phosphorylation of opsin and three proteins of 48, 68, and 200 kDa, as well as a light-dependent reversible binding of the 48-kDa protein to the rhabdomal membranes (16). In none of these studies, however, have the functional consequences ofthese reactions been determined. Two ofthe Drosophila phosphoproteins, those of 39 and 49 kDa, have been identified as arrestin homologues by molecular cloning (17)(18)(19)(20), suggesting that they may pl...