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Cells transformed by Kirsten murine sarcoma virus (Ki-MSV) have basal adenylate cyclase activity (AC) higher than control cells and comparable level of forskolin-stimulated AC activity. Moreover, a higher protein kinase C (PKC) activity was found to be present in the transformed cells. The molecular mechanism underlying the increase of AC activity was investigated. Our findings strongly suggest that this biochemical event is due to a marked decrease of the α i negative control of the enzyme, even though the α i of transformed cells appears to possess fully functional domains interacting with both the effector enzyme and the agonist-activated receptor.

Background : cAMP is a second messenger that plays a role in intracellular signal transduction of various stimuli. a major function of cAMP in eukaryotes is activation of cAMP-dependent protein kinase (PKA). PKA is the best understood member of the serine-threonine protein kinase superfamily, and is involved in the control of a variety of cellular processes. since it has been implicated in the initiation and progression of many tumors, PKA has been suggested as a novel molecular target for cancer therapy. Objective/methods : here, after describing some features of cAMP/PKA signaling that are relevant to cancer biology, we review targeting of PKA in cancer therapy, also discussing PKA as a biomarker for cancer detection and monitoring of therapy. Results/conclusions : PKA is an increasingly relevant biological target in the therapy and management of cancer.

In order to elucidate how phosphate regulates cellular functions, we investigated the effects of inorganic phosphate (Pi) on adenylate cyclase (AC)/cyclic AMP (cAMP) axis. Here we describe that Pi treatment of human osteosarcoma U2OS cells results in a decrease of both intracellular cAMP levels and AC activity, and in a cell growth inhibition. The phosphate-triggered effects observed in U2OS cells are not a widespread phenomenon regarding all cell lines, since other cell lines screened respond differently to parallel Pi treatments. In U2OS cell line, the AC activity/cAMP downregulation is accompanied by significant variations in the levels of some membrane proteins belonging to the AC system. Remarkably, the above effects are blunted by pharmacological inhibition of sodium-dependent phosphate transport. Moreover, 8-Br-cAMP and other cAMP-elevating agents, such as IBMX and forskolin, interestingly, prevent the cell growth inhibition in response to phosphate. Our results enforce the increasing evidences of phosphate as a signaling molecule, identifying in U2OS cell line the AC/cAMP axis, as a novel-signaling pathway modulated by phosphate to ultimately affect cell growth.

We have found that in the secretion of rat anterior prostate, a hydrolyzing activity on GTP is present with a high affinity for the substrate; ATP, GDP, and ADP are not substrates for enzymatic activity. At the same time we have shown that GTP is a negative modulator for the well-known type IV transglutaminase activity present in the prostatic secretion. The hydrolyzing activity on GTP appears to be due to two molecular species: a high-molecular-weight GTPase, having electrophoretical mobility higher than 100 kDa, and a low-molecular-weight GTPase, of about 30 kDa. The two enzymatic activities are associated in the prostatic secretion with the transglutaminase (type IV). We describe an experimental procedure to separate them.

We have investigated the in vitro phosphorylation of high molecular weight glutenin subunits (HMW-GS), a group of non-soluble proteins present in wheat endosperm. Computer aided searches of potential biological sites in the known sequences of these proteins have evidenced the presence of sequence motifs specific for protein kinase C (PKC), calcium-dependent protein kinase from wheat, casein kinase II, tyrosine protein kinase and glycosylation. We have demonstrated that subunit 1Bx7 is a substrate of a partially purified PKC from rat brain. Further experiments have shown that this subunit is phosphorylated by an endogenous protein kinase activity found in wheat flour. These preliminary results are important for the possible implications on the structure-function relationships of these proteins and could probably suggest, for the first time, a potential physiological role in particular situations for some HMW-GS.

Both cytoplasmic and membrane-bound protein kinase C activities are increased in: Harvey-Sarcoma Virus, infected thyroid epithelial cells. The cytoplasmic kinase C increase is found in the chromatographic fraction eluted at lower salt concentration (100 mM NaCl-S100), while the more acidic protein fraction eluted at higher salt concentration (350 mM NaCl-S350) is virtually absent. Although the cytoplasmic S100 fraction from the control and ras-virus infected cells display a comparable PBt2 binding activity, they are different in the Ca+2-dependence and the TPA down regulation. In addition, the membranes from the control and ras-virus infected cells are different phosphate acceptors in place of the Hl histones. © 1988 Academic Press, Inc.

The soluble guanylate cyclase activity of rat liver appears to be stimulated in VITRO by insulin at pMolar concentrations, while proinsulin, denaturated insulin or desoctapeptide insulin, are not able to stimulate the studied enzymic activity. Corresponding concentrations of other peptide hormones such as corticotropin (ACTH) or glucagon, either in the absence or in the presence of bacitracin, do not show any effect on the investigated enzymic system. Insulin stimulation of the soluble guanylate cyclase is characterized by a significant increase in the Vmax together with a decrease of the appearent Km. Insulin at low concentrations doesn't affect the cyclic GMP hydrolyzing activity; conversely higher concentrations of the hormone, while exerting a less marked effect on the guanylate cyclase activity, inhibit the cyclic GMP hydrolyzing activity.

The high affinity (low Km) cyclic GMP phosphodiesterase (PDE) is activated by GTP, while the cyclic AMP PDE is not. GTP and its hydrolysis-resistant analogue, guanylylimidodiphosphate (GppNHp), display a half-maximal stimulating effect at almost the same concentration (5 × 10−6M). The GTP stimulating effect is not observed when the socalled cyclic GMP low affinity (high Km) PDE is operative. GTP cooperates with the increase of the substrate concentration on removing the IBMX inhibitory effect. The isolation through a classical chromatographic procedure on a DEAE-cellulose column, of a PDE fraction specific for cyclic GMP, results in the loss of the GTP stimulating effect.

High protein dietary content stimulates urea formation in ureotelic animals but does not exert almost any effect on ammonia production from L-amino acids in vitro. L-histidine and L-threonine are the only amino acids which are most actively deaminated by ureotelic animals fed on a high protein diet. All the steps of L-histidine metabolism have been studied: it has been found that both the histidine transaminase pathway and the histidase pathway are stimulated. Glutamic acid is also a product of histidine catabolism through the histidase pathway, but its catabolism is unaffected by the dietary protein content. These data suggest the existence of independent mechanism controlling the catabolism of the two amino acids.

1. 1. The effects of polyamines spermine, spermidine and putrescine have been investigated on the adenylate cyclase and phosphodiesterase activities bound to E. coli B-cell membranes. 2. 2. Putrescine, which is physiologically present in bacteria, affects the cAMP metabolism in a coordinate manner, depressing the adenylate cyclase and stimulating the phosphodiesterase activities. In vivo this is reflected by a decreased cAMP efflux toward the external medium. 3. 3. Spermidine stimulates the phosphodiesterase activities but has no effect on the adenylate cyclase; furthermore no effect was observed in vivo on the cAMP efflux. li[4. Spermine stimulates both the enzymic activities but with a prevailing effect on the adenylate cyclase. In vivo the effect is an increase in the cAMP efflux. 4. 5. In vivo the intracellular cAMP content is anyway not modified in presence of polyamines, in contrast with (and may be because of) the broad variation of the nucleotide efflux toward the external medium.

Escherichia coli endotoxin inhibited the fluoride-stimulated adenylate cyclase activity of liver plasma membranes enriched with bile canaliculi. Inhibition was of a mixed competitive and uncompetitive type. This effect, which may result from changes of membrane organization, may have relevance in the understanding of endotoxin-cell membrane interactions.

Blood plasma levels of S -adenosylmethionine have been measured in humans by a recently developed isotope dilution technique. The procedure involves isolation of the sulfonium compound by ion-exchange chromatography after its extraction with perchloric acid. Data are presented on the blood plasma content of S -adenosylmethionine in humans at different ages; the levels of the compound seem to be related to the age.

β-METHYLASPARTATE (β-MA) was identified1 as a product of glutamate metabolism in cell-free extracts of Clostridium tetanomorphum. β-MA is a strong antimeta-bolite toward aspartate: it is an inhibitor of pyrimidine biosynthesis from aspartate2 and of urea synthesis from citrulline + aspartate3. In addition, β-MA seems to have certain interesting metabolic activities: it participates in transamination reactions in rat and rabbit liver and brain4 and in Escherichia coli5, and it may also be a precursor in the de novo synthesis of thimine6.