Carmen Bordallo

Localization of the thermosensitive X-prolyl dipeptidyl aminopeptidase in the vacuolar membrane of Saccharomyces cerevisiae

Most of the X‐prolyl dipeptidyl aminopeptidase activity of Saccharomyces cerevisiae was found to be associated with purified vacuolar membranes (specific activity approx. 75‐times higher than in the protoplast lysate). The tonoplast‐bound enzyme is thermosensitive. Another heat‐resistant enzyme was found in the protoplast lysate. The tonoplast‐bound thermosensitive enzyme shows an apparent K m of 0.06 mM against L‐alanyl‐L‐prolyl‐p‐nitroanilide while the heat‐resistant enzyme shows an apparent K m of 0.4 mM against the same substrate.

Modulatory role of endogenous androgens on airway smooth muscle tone in isolated guinea-pig and bovine trachea; involvement of β2-adrenoceptors, the polyamine system and external calcium

Androgens relax several smooth muscles, including the airways. They also contract ileum and myocardium via nongenomic mechanisms. To find out whether androgens modulate airway smooth muscles in different species and further assess their mechanism of action, regarding the role of beta-adrenoceptors, polyamines and extracellular Ca(2+), and the modulation of contraction, 5 alpha-dihydrotestosterone, testosterone and 5 beta-dihydrotestosterone were used. A preliminary study was performed to evaluate the effect of 5 alpha-dihydrotestosterone, a non-aromatisable derivate of testosterone, in isolated guinea-pig trachea and a more exhaustive characterisation was followed in bovine trachea, to also characterise the effect of testosterone and 5 beta-dihydrotestosterone. The androgens elicited a nongenomic epithelium-independent relaxation of the trachea which had been precontracted. In the bovine trachea, the order of potency was: testosterone>5 alpha-dihydrotestosterone=5 beta-dihydrotestosterone. This effect was inversely proportional to the magnitude of carbachol-raised tone and was independent of beta(2)-adrenoceptors, since the beta-blockers, propranolol and ICI-118,551, and beta(2)-adrenoceptor desensitisation did not modify 5 alpha-dihydrotestosterone-elicited relaxation. 5 alpha-Dihydrotestosterone was unable to displace the radiolabel, [(3)H]dihydroalprenolol, from these receptors in the binding assay. Polyamine synthesis was not involved in this androgen effect, since an ornithine decarboxylase inhibitor, alpha-difluoromethylornithine, was ineffective. The androgens were more effective relaxing bovine trachea precontracted by KCl (80 mM), suggesting a calcium entry blockade, as reported for several smooth muscles. This mechanism might be involved in the observed 5 alpha-dihydrotestosterone facilitation of salbutamol-relaxation. Androgens facilitated carbachol-elicited contraction independently of polyamine synthesis, contrary to what has been reported in the ileum. Therefore, androgens modulate tracheal smooth muscle tone which might be of importance in the regulation of airway reactivity.

Increases in ornithine decarboxylase activity in the positive inotropism induced by androgens in isolated left atrium of the rat

It is well established that the intracellular receptors of androgens act as transcription factors upon their activation by androgen binding. However, a growing number of studies have associated androgens with rapid biological responses independent of their classical action mechanism. In this sense, 5alpha- and 5beta-dihydrotestosterone elicited a rapid positive inotropism in the isolated left atrium of the rat via cAMP-dependent mechanisms that may involve genomic effects. In addition, polyamines are mediators of several biological actions including those acute and long-term effects induced by androgens in the heart. The present study analyzed the role of polyamine synthesis in the cardiotonic effect of androgens in the left atrium of male Wistar rats, electrically stimulated (0.5 Hz, 5 ms and supramaximal voltage) and placed in an organ bath in 10 ml of Tyrodes solution. Incubation in the organ bath with an inhibitor of ornithine decarboxylase activity, alpha-difluoromethylornithine 10 mM, significantly decreased the positive inotropism induced by 5alpha- and 5beta-dihydrotestosterone (0.1-100 microM). This suggests that ornithine decarboxylase seems to be involved in androgen-induced positive inotropism. Furthermore, 6-min exposure to 5alpha- or 5beta-dihydrotestosterone significantly increased the activity of ornithine decarboxylase from 61.81+/-7.53 (control) to 93.28+/-9.45 and 80.28+/-12 pmol/h/mg of protein, respectively. Northern blot analysis showed that 5alpha- and 5beta-dihydrotestosterone did not modify the level of expression of the ornithine decarboxylase gene. Therefore, our results suggest that polyamine synthesis might be involved in the positive inotropism elicited by androgens through the stimulation of ornithine decarboxylase activity without changes in the expression of the ornithine decarboxylase gene.

POSITIVE INOTROPISM INDUCED BY ANDROGENS IN ISOLATED LEFT ATRIUM OF RAT : EVIDENCE FOR A CAMP-DEPENDENT TRANSCRIPTIONAL MECHANISM

Steroid hormones exert their biological actions via intracellular receptors modulation of transcription. In addition, a number of molecular interactions, and the existence of membrane receptors in several tissues, support the hypothesis of nongenomic action of steroids. The androgens, 5alpha- and 5beta-dihydrotestosterone (0.1 to 100 microM), induce a rapid positive inotropism in the isolated left atrium of male Wistar rats whose time course of response might suggest that it is a non-genomic effect. However, the fact that the facilitation of contractility was inhibited by actinomycin D (5 microg/ml) and cycloheximide (10 microg/ml) indicates that a transcriptional component might play a role. The existence of a rapid functional genomic role would be somewhat surprising. However, rapid transcriptional mechanisms were also observed in certain cAMP-dependent responses. In the left atrium of rat, Rp-cAMPS (10 microM), a cAMP-dependent protein kinase inhibitor, antagonized 5alpha- but not 5beta-dihydrotestosterone-induced positive inotropism. The inhibition by Rp-cAMPS of isoproterenol- and forskolin-induced positive inotropism, and the fact that these cAMP-dependent effects were also inhibited by actinomycin D and cycloheximide, suggest that a cAMP-dependent transcriptional component may be partly involved in the positive inotropism induced by 5alpha-dihydrotestosterone. In addition, 5alpha-dihydrotestosterone might increase the basal adenylyl cyclase activity by acting on unoccupied beta-adrenoceptor-G-protein-adenylyl cyclase complexes, since the elicited inotropism was inhibited by a beta-blocker, atenolol (1 microM), a G-protein inhibitor, pertussis toxin (2 microg/ml, 3 h), and an adenylyl cyclase inhibitor, dideoxy-adenosine (10 microM).

Molecular cloning and sequencing of genomic DNA encoding yeast vacuolar carboxypeptidase yscS

A Saccharomyces cerevisiae genomic DNA encoding vacuolar carboxypeptidase yscS was cloned from a yeast YEp 13 library by complementation of the previously characterized mutation epsl‐1 [(1981) J. Bacteriol. 147, 418–426], by means of staining carboxypeptidase activity in yeast colonies. The nucleotide sequence of the cloned gene was determined. The open reading frame of CPS1 consists of 576 codons and therefore encodes a protein of 64961 molecular weight. A stretch of 19 residues near the N‐terminus of the deduced polypeptide sequence contains characteristics common to known hydrophobic leader sequences. CPS1 was determined by DNA blot analysis to be a single copy gene located on chromosome X. The cloned fragment was used to identify a 2.1 kb mRNA. A transcriptional activation of CPS1 occurs when cells grow on a substrate of carboxypeptidase yscS as sole nitrogen source.

Purification and characterization of a thermosensitive X-prolyl dipeptidyl aminopeptidase (dipeptidyl aminopeptidase yscV) from Saccharomyces cerevisiae

Abstract Dipeptidyl aminopeptidase yscV, a heat-labile enzyme with X-prolyl dipeptidyl aminopeptidase activity, was purified about 470-fold from a protoplast lysate of Saccharomyces cerevisiae. The purification procedure included solubilization of tonoplast-bound activity by the non-ionic detergent octyl-β- d -glucopyranoside, glycerol gradient centrifugation and preparative isoelectric focusing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis resulted in a single band for which a molecular weight of 40 000 was calculated. The peptidase was most active at pH 7.0–7.5 with l -alanyl- l -proline -p- nitroanilide as substrate. Substrate specificity studies indicate that the purified enzyme specifically hydrolyzes peptide bonds involving the carboxyl group of prolyl residues penultimate to unprotected termini unless arginine is the N-terminal amino acid. However, X-Ala-arylamide structures are not attacked. The actinomycete inhibitors antipain, chymostatin and pepstatin had no effect on the enzyme activity, but 5 mM phenylmethylsulfonyl fluoride, an inhibitor of serine peptidases, completely inhibited dipeptidyl aminopeptidase yscV activity. Some heavy metals (Ni2+, Cd2+, Zn2+, Hg2+) at a concentration of 5·10−4 M were also found to be potent inhibitors of enzyme activity.

Intracellular cAMP increases during the positive inotropism induced by androgens in isolated left atrium of rat

Molecular interactions of androgens with the plasma membrane may produce rapid cardiovascular effects that cannot be explained by the classic genomic mechanisms. In this sense, 5 alpha- and 5 beta-dihydrotestosterone-induced an acute positive inotropic effect in isolated left atrium of rat, an effect which may be due to cAMP-dependent mechanisms. To prove this, intracellular levels of cAMP, after exposure to androgens in the organ bath, and binding to beta(1)-adrenoceptors were evaluated. After a 4-min exposure, 5 alpha- and 5 beta-dihydrotestosterone increased cAMP levels from 3.83+/-0.61 to 6.15+/-1.1 and 11.18+/-2.4 pmol cAMP/mg of protein, respectively. These increases were inhibited by atenolol and not modified by treatment of the rats with reserpine. The androgen-induced cAMP increase seems to be produced via an extracellular interaction, because positive inotropism and raised levels of cAMP were produced by 5 alpha-dihydrotestosterone conjugated with bovine serum albumin (BSA). In addition, it is independent of beta(1)-adrenoceptor activation, because neither androgen displaced [(3)H]dihydroalprenolol binding. Therefore, the androgens induced a positive inotropic effect via a postsynaptic effect that increases intracellular levels of cAMP. This effect is modulated by transcriptional mechanisms or by a protein with a short half-life.

Putrescine modulation of acute activation of the β-adrenergic system in the left atrium of rat

Endogenous polyamines mediate acute metabolic effects and cardiac hypertrophy associated to beta-adrenoceptor stimulation. The aim of this study is to characterize the role of polyamines on beta-adrenoceptor system mediated responses. To this end, the functional interaction of polyamine modifying drugs on isoproterenol-elicited cardiotonic effect, in isolated left atria of male Wistar rats, and their effects on [(3)H]dihydroalprenolol (DHA) binding on beta-adrenoceptors and on adenylyl cyclase activity of membrane heart were studied. Polyamines interact with beta-adrenoceptors in rat heart, as shown by the displacement of [(3)H]DHA binding. Furthermore, putrescine (but not spermidine or spermine) increased adenylyl cyclase activity, elicited a positive inotropism and increased intracellular cAMP. The putrescine effect on adenylyl cyclase was not antagonized by the beta-adrenoceptors blockers, alprenolol and ICI-118,551, and facilitated the isoproterenol effect. Neither alprenolol, atenolol nor ICI-118,551 antagonized putrescine-elicited positive inotropism. However, the effect was abolished in preparations with desensitized beta-adrenoceptors. alpha-Difluoromethylornithine, an inhibitor of ornithine decarboxylase, antagonized the effect of isoproterenol on inotropism and cAMP increase. In addition, putrescine might elicit effects by mechanisms independent of beta-adrenoceptor system, since in left atria with functional desensitized receptors an interaction with ouabain-elicited cardiotonic effect was observed. These results suggest that putrescine may act as a low affinity agonist on beta-adrenoceptors and modulate acute responses mediated by beta-adrenoceptors. These findings may be of importance in the physiology and in diseases involving cardiac beta-adrenoceptors.

Role of putrescine on androgen-elicited positive inotropism in the left atrium of rats.

Functional and biochemical studies were performed in isolated left atria of male Wistar rats to study whether endogenous polyamines may mediate androgen-elicited positive inotropism and their relationship with a rise in cAMP during the cardiotonic effect. 5α-Dihydrotestosterone (100 μM) exposure increased intracellular putrescine as determined by HPLC, but it did not increase spermidine and spermine. This effect was antagonized by an inhibitor of ornithine decarboxylase, α-difluoromethylornithine (10 mM), suggesting enzyme activation. α-Difluoromethylornithine also antagonized androgens-elicited inotropism and the increase in intracellular cAMP. Putrescine (1 to 10 mM) elicited a concentration-dependent positive inotropism associated with the cAMP increase. The prior incubation with putrescine antagonized 5α-dihydrotestosterone-elicited inotropism and did not produce sinergism on intracellular cAMP. Short-term incubation with 5α-dihydrotestosterone or forskolin shifted to the left the cardiotonic effect of isoproterenol, an agonist of β-adrenoceptors, without any increase in Emax, suggesting that a common mechanism was involved. Therefore, polyamines might modulate the cAMP production associated with the cardiotonic effect of androgens.

Localization of Dipeptidyl Aminopeptidase yscIV in the Plasma Membrane of Saccharomyces Cerevisiae

The subcellular distribution of dipeptidyl aminopeptidase activity was studied in protoplast lysates of Saccharomyces cerevisiae that were virtually free from vacuolar contamination. Dipeptidyl aminopeptidase yscIV, the STE13 gene product, was found to be associated with plasma membrane vesicles after sucrose gradient isopycnic centrifugation. Another dipeptidyl aminopeptidase activity, not yet fully characterized, was localized in a microvesicular population co-sedimenting with chitosomes.