Activity Of Membrane-associated Enzymes Research Articles

Use of Azoles for Systemic Antifungal Therapy

The azole antifungal agents have expanded the armamentarium of agents useful in the treatment of systemic fungal infections. Clotrimazole, an early imidazole antifungal, proved was found to rapidly induce its own metabolism after oral or intravenous administration. Its use is now confined to topical therapy. Miconazole is toxic and its availability is only as an intravenous formulation. Ketoconazole, the first oral imidazole, has proved useful for a wide variety of fungal infections. This chapter focuses on the use of these four azole agents that have proved beneficial in treating systemic fungal infections. The imidazole compounds contain a five member azole ring with two nitrogen atoms. The triazoles have a third nitrogen atom added. The triazole ring increases tissue penetration, prolongs half-life, and enhances efficacy of the azoles while decreasing the toxicity when compared with imidazole antifungal agents. Azoles inhibit the biosynthesis of ergosterol, a component of fungal cell membranes, via inhibition of the cytochromes P450-dependent enzyme lanosterol14-α-demethylase.Lanosterol14-α-demethylase is necessary for the conversion of lanosterol to ergosterol. Depletion of ergosterol in the fungal cell membrane results in altered membrane fluidity, thereby reducing the activity of membrane-associated enzymes. This leads to increased permeability and subsequent inhibition of cell growth and replication. The binding efficiency of azole antifungal agents to cytochromes P450 differs, resulting in differences in antifungal activity, toxicity of the agents, and the drug interactions with other cytochrome P45O-metabolized drugs. The chapter details the antifungal activity of the azoles, their pharmacology, clinical use, side effects, and administration. Drug interactions with azole antifungals are of two categories—decreases in azole bioavailability because of chelation or secondary to increases in gastric pH; and interactions with other cytochrome P45O-metabolized drugs. Drug interactions in the second category may result in increases or decreases in the azole antifungal, in the interacting drug, or in both drugs.

Suppression of cornified envelope formation and type 1 transglutaminase by epidermal growth factor in neoplastic keratinocytes.

Epidermal growth factor (EGF) is a potent mitogen for keratinocytes. Although the role of the EGF receptor in cell proliferation has been extensively studied, the consequences of EGF receptor activation with respect to cell differentiation remain less well characterized. Our studies demonstrate that stimulation of the EGF receptor substantially suppresses cellular differentiation in squamous cell carcinoma lines that overexpress the EGF receptor, as assessed by an EGF-dependent reduction of cornified envelope formation. Only a modest ligand-dependent decrease in cornified envelope formation was observed in normal keratinocytes. The response is dependent on the concentration of EGF and is evident after 1-2 days of EGF treatment. With extended EGF treatment, the messenger RNA levels for involucrin, a major structural component of the cornified envelope, were unaltered by EGF. In contrast, membrane-associated transglutaminase enzyme activity, which predominantly represents type 1 (keratinocyte) transglutaminase, is markedly inhibited by EGF. The lost of type 1 transglutaminase activity is associated with reduced levels of the messenger RNA and protein. These studies suggest that the functional consequences of EGF receptor activation in squamous cell carcinomas involve not only aberrant growth regulation, but, additionally, reduction of terminal differentiation capacity.

Suppression of cornified envelope formation and type 1 transglutaminase by epidermal growth factor in neoplastic keratinocytes

Epidermal growth factor (EGF) is a potent mitogen for keratinocytes. Although the role of the EGF receptor in cell proliferation has been extensively studied, the consequences of EGF receptor activation with respect to cell differentiation remain less well characterized. Our studies demonstrate that stimulation of the EGF receptor substantially suppresses cellular differentiation in squamous cell carcinoma lines that overexpress the EGF receptor, as assessed by an EGF-dependent reduction of cornified envelope formation. Only a modest ligand-dependent decrease in cornified envelope formation was observed in normal keratinocytes. The response is dependent on the concentration of EGF and is evident after 1-2 days of EGF treatment. With extended EGF treatment, the messenger RNA levels for involucrin, a major structural component of the cornified envelope, were unaltered by EGF. In contrast, membrane-associated transglutaminase enzyme activity, which predominantly represents type 1 (keratinocyte) transglutaminase, is markedly inhibited by EGF. The lost of type 1 transglutaminase activity is associated with reduced levels of the messenger RNA and protein. These studies suggest that the functional consequences of EGF receptor activation in squamous cell carcinomas involve not only aberrant growth regulation, but, additionally, reduction of terminal differentiation capacity.

Estrogen increases nitric oxide synthase activity in the uterus of nonpregnant sheep

OBJECTIVE: The aim of this study was to characterize nitric oxide synthase activity in endometrium and myometrium of nonpregnant sheep and to determine whether estrogen administration affects uterine nitric oxide synthase activity. STUDY DESIGN: Nonpregnant sheep were castrated during synchronized estrus and 4 days after surgery were treated with 100 μg/day of 17β-estradiol for 3 days. Nitric oxide synthase activity was measured by the citrulline conversion assay. RESULTS: Citrulline generation found in soluble and particulate fractions had all the characteristics of nitric oxide synthase, namely, it was strictly dependent on reduced nicotinamide adenine dinucleotide phosphate and enhanced by flavin nucleotides and tetrahydrobiopterin. Estrogen administration significantly increased Ca ++-dependent nitric oxide synthase activity in myometrium but not in endometrium. The effect was more pronounced in the membrane-associated enzyme activity (approximately fivefold). Estrogen treatment increased myometrial nitric oxide synthase activity from 9.0 ± 2.4 to 20.0 ± 3.7 pmol/mg of protein per 30 minutes in the soluble fraction and from 12.0 ± 5.1 to 62.0 ± 13.1 pmol/mg of protein per 30 minutes in the particulate fraction (mean ± SEM, p < 0.05 by t test). The increase in nitric oxide synthase activity was not mediated by an increase in tetrahydrobiopterin availability, as shown to be the case in macrophages. CONCLUSION: These data show that in the nonpregnant sheep uterus > 90% of the nitric oxide synthase activity found in myometrium is Ca ++ dependent and is up-regulated by estrogen in a tissue-specific manner.

Effect of a high alpha-linolenate and high linoleate diet on membrane-associated enzyme activities in rat brain--modulation of Na+, K+- ATPase activity at suboptimal concentrations of ATP.

Semi-purified diets supplemented with either a high alpha-linolenate (n - 3) (perilla) oil or a high linoleate (n - 6) (safflower) oil were fed to rats through two generations. Rats fed safflower oil showed a decrease in docosahexaenoic acid (n - 3) and a compensatory increase in docosapentaenoic acid (n - 6) in all the brain regions and organelles examined, when compared with rats fed perilla oil. As reported previously, the safflower oil-fed rats exhibited inferior learning ability compared with the perilla oil-fed rats (N. Yamamoto et al., J. Lipid Res. 28, 144 (1987)). Using brains of rats in these dietary groups, the activities of several enzymes, Na+ , K+-ATPase, Ca2+-ATPase, 5'-nucleotidase, 2',3'-cyclic nucleotide phosphodiesterase, acetylcholinesterase, and choline acetyltransferase in membranes, were compared. The 5'-nucleotidase activity in cortex and hippocampus, and the Na+, K+-ATPase activity in myelin decreased slightly but significantly in the safflower oil group. None of the other membrane-associated enzyme activities in all the brain regions and organelles examined was affected significantly by the dietary fatty acids under optimal assay conditions in vitro. However, in the safflower oil group, the Na+, K+-ATPase activity of synaptosomes at a suboptimal concentration of ATP was 78% that in the perilla oil group. These results suggest that relatively large changes in the proportions of n - 3 and n - 6 polyunsaturated fatty acids in brain membranes caused by dietary manipulation do not provoke significant alterations in most membrane-bound enzyme activities. However, a small but significant change in Na+, K+-ATPase activity at a suboptimal concentration of ATP may be implicated in the altered learning behavior reported earlier.

Hyperpolarization-relaxation coupling in vascular smooth muscle

Studies performed to elucidate the inhibitory mechanisms of the hyperpolarization induced by K+ channel openers on the Ca2+ movements and force of contraction produced by either the stimulation of thromboxane A2 receptors or depolarization with high KCl have shown the following: When the plasma membrane is hyperpolarized by K+ channel openers, voltage-dependent L-type Ca2+ channels are deactivated and the influx of Ca2+ is decreased, as is the case with the KCl-induced Ca2+ influx. The hyperpolarization of the plasma membrane also has other inhibitory effects on phospholipase C, a membrane-associated enzyme activity. The IP3 production and IP3-induced Ca2+ release from intracellular stores, which is related to the stimulation of the agonist receptors, are inhibited by the hyperpolarization of the plasma membrane by K+ channel openers. Recently, we showed that the Ca2+ sensitivity of the contractile elements was voltage dependent. Furthermore, membrane hyperpolarization induced by various K+ channel openers relaxed canine coronary arteries more profoundly than decreased [Ca2+]i. Thus, the membrane voltage may regulate intracellular enzyme activities, including contractile elements. Therefore, this new facet of signal transduction should be considered in the control of vascular tone. The evolutional relationships of various K+ channels are also discussed.

Ethanol and protein kinase C in rat brain

The effect of chronic ethanol consumption on the catalytic activity of protein kinase C isolated from rat brain was studied in two different ways. Enzyme activity was first measured by phosphorylation of Histone IIIS in vitro. There was no change in the activity of the cytosolic enzyme. Membrane-associated enzyme activity was reduced in the ethanol-treated animal. This difference was not evident if the enzyme was stimulated by arachidonate. The reduction in enzyme activity was confirmed by analysis of the phosphorylation of endogenous substrates in intact synaptosomes. When the binding of the ligand [ 3H]phorbol dibutyrate was measured by quantitative autoradiography, increased binding to membrane-associated protein kinase C was observed in the CA1 region of the hippocampus but not in other brain regions. These results indicate that ethanol treatment results in a general reduction in membrane-associated protein kinase C activity as measured in vitro but the effect may not be consistent in all brain regions. The differential effect in the CA1 region of the hippocampus may be a reflection of a disruption in the normal regulation of protein kinase C activity in this area and may indicate that this region is a sensitive target for the action of ethanol.

Oxysterol-induced endothelial cell dysfunction in culture.

Cholesterol oxidation products (oxysterols), such as cholestan-3 beta,5 alpha,6 beta-triol (Triol), may be atherogenic by altering the barrier function of the vascular endothelium. We have shown that incubation of endothelial cell monolayers with Triol increased transendothelial albumin transfer (i.e., decreased barrier function) in a concentration- and time-dependent manner. Such dysfunction of endothelium could result from alterations in membrane characteristics, including changes in membrane-associated enzyme activities. To test this hypothesis, endothelial monolayers were treated with 20 microM Triol and the activities of selected membrane enzymes were measured at 0, 2, 4, 6, 12 and 24 hours. Calcium-adenosine triphosphatase (Ca(++)-ATPase) and sodium, potassium, magnesium-adenosine triphosphatase (Na+, K+, Mg(++)-ATPase) activities were significantly increased after 4 or 2 hours incubation with 20 microM Triol, respectively. 5'-nucleotidase activity was significantly elevated only after a 24-hour exposure to Triol, whereas there was no change in angiotensin-converting enzyme (ACE) activity in response to 20 microM Triol treatment at any time studied. Compared with all concentrations tested 40 microM Triol increased Ca(++)-ATPase activity most markedly, with a significant increase already after a 2-hour exposure. No major morphological changes were noted until 12 hours of exposure to 20 microM Triol; obvious cellular damage was observed by 24 hours. Cultures treated with Triol for 24 hours showed significant signs of toxicity, measured by an elevated [3H]adenine release, compared with control cultures. These data demonstrate that Triol alters the activity of certain membrane-bound enzymes, particularly Na+, K+, Mg(++)-ATPase and Ca(++)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of phospholipase A2 activation and arachidonic acid metabolism in an interleukin-3-dependent macrophage-like cell line.

An interleukin 3 (IL-3)-dependent macrophage-like cell line, 11-1-B3, was newly established from CBA/J mouse bone marrow cell cultures. Assay of eicosanoids in the culture supernatants of the intact and [3H]arachidonic acid (AA)-prelabeled cells showed that, after stimulation with the Ca2+ ionophore A23187, the 11-1-B3 cells synthesized and released relatively large amounts of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) but not LTC4. In addition, 11-1-B3 cells showed Ca(2+)-dependent and alkaline pH-optimal phospholipase A2 (PLA2) activity that preferentially hydrolyzed cleavage of sn-2-arachidonyl- but not sn-2-oleoylphosphatidylcholine. The cellular enzyme was distributed with 90% of the activity in the cytosol and 10% in the membrane fraction. Treatment of cells with A23187 for 5-10 min resulted in five- to sevenfold increases in the membrane-associated PLA2 but activity in the cytosol was unchanged. This increase in membrane-associated enzyme activity was transient, returning to the pretreatment distribution after 30 min. In sharp contrast, phorbol myristate acetate (PMA) stimulation failed to induce either eicosanoid release or PLA2 activation, although PMA induced translocation of protein kinase C (PKC) to the membrane fraction within 10 min. The data suggest that increases in cellular Ca2+ directly activate membrane-associated PLA2 and consequently initiate AA metabolism; PKC activation by PMA requires additional steps to activate PLA2, a mechanism that is apparently deficient in the IL-3-dependent M phi-like cells.

The effect of UV radiation and sun blockers on free radical defence in human and guinea pig epidermis

Defence against oxidative damage by UV-generated free radicals in both guinea pig and human skin has been found to be mediated by the ubiquitous thioprotein, thioredoxin reductase. Human keratinocytes contain approximately 5% thioredoxin reductase in their total acidic protein fraction and also express membrane-associated enzyme activity in cells cultured in synthetic medium. The thioredoxin reductase/thioredoxin system has been shown to reduce superoxide anion radicals through hydrogen peroxide to water. However, both UVA and UVB radiation, below the minimal erythemal dose, generate a sufficiently high concentration of oxygen radicals to deactivate thioredoxin reductase considerably. In albino guinea pigs, enzyme deactivation was up to 70% for UVA and 66% for UVB (n = 10 animals/protocol). The application of sun blockers SPF4, SPF8 and SPF15 to albino guinea pig skin offered no significant protection for the deactivation of thioredoxin reductase by either UVA or UVB radiation. In the human population (n = 15), thioredoxin reductase was deactivated by 54% with UVA and 36% with UVB radiation, although the degree of enzyme inhibition depended on skin phototype (I-VI, Fitzpatrick Classification). SPF24 offered considerable protection for thioredoxin reductase against both UVA and UVB for skin types I and II. However, SPF24 yielded no significant protection with UVA for skin types III-VI, and enhanced the enzyme inhibition with UVB additively. These results indicate that UVB photo-oxidation of oxybenzone (the UVA filter in SPF24) may deactivate thioredoxin reductase in more pigmented members of the population by Michael addition of oxybenzone semiquinone to the thiolate active site of this enzyme.

The Response of Living Cells to Very Weak Electric Fields: the Thermal Noise Limit

A physical model in which cells are considered as possible detectors of very weak periodic electric fields yields a general relation between cell size and both thermally induced fluctuations in membrane potential and the maximum change in membrane potential caused by an applied field. The simplest version of the model provides a broad-band estimate of the smallest applied electric field to which membrane macromolecules can directly respond (about 10(-3) volt per centimeter). Much smaller fields (10(-6) volt per centimeter) can be detected if there is a response in only a narrow band of frequencies or if signal averaging occurs through field-induced variation in the catalytic activity of membrane-associated enzymes. Both extensions of the simplest version remove the apparent violation of the thermal noise limit found in some experiments.

Characterization of cytoplasmic and membrane-associated phosphatidylinositol 4,5-biphosphate phospholipase C activities in guinea pig ventricles

The phosphoinositide-specific phospholipase C (PLC) activity present in the soluble and sarcolemmal enriched membrane fraction from guinea pig hearts was characterized using phosphatidyl [3H]inositol 4,5-biphosphate (PIP2) or phosphatidyl [3H]inositol 4-monophosphate (PIP) as substrates. The PLC activities (cytosolic and membrane associated) were specific for polyphosphoinositides (PIP2 and PIP) since no other phospholipids were hydrolyzed at pH 7.0 under various ionic conditions. Both enzymic activities were Ca2(+)-dependent (half maximal activities were achieved around pCa 5.0). The pH, detergent (deoxycholate), divalent (Ca2+ and Mg2+), and monovalent (Na+ and K+) cation dependencies were very similar between the cytosolic and membrane-associated enzyme activities, using either PIP2 or PIP as substrate. Hydrolysis of the polyphosphoinositides was inhibited in the presence of phosphatidylethanolamine, phosphatidylserine, or phosphatidylcholine. Under optimal conditions (pH 7.0, 1 mM Ca2+, 2.5 mM Mg2+, 100 mM Na+ and 0.07% deoxycholate) the specific activities of the cytosolic and membrane-associated enzymes were 19.9 +/- 0.9 and 10.1 +/- 0.9 nmol/min/mg protein, respectively, using PIP2 as substrate. Under the same conditions these activities were 18.1 +/- 1.0 and 8.0 +/- 0.8 nmol/min/mg protein for the cytosolic and membrane fractions, respectively, using PIP as substrate. Based on the similarity of the characteristics of these two PLC enzyme activities, it is suggested that the cytosolic and membrane-associated enzyme forms may be closely related.

Membrane fluidity and cancer metastasis.

The membrane fluidity of murine B16 melanoma and L5178 lymphoma variants is examined in relation to their metastatic potential. A higher lateral mobility of membrane proteins in metastasis is indicated by lectin receptor-mediated agglutination studies, but these do not constitute incontrovertible evidence that higher fluidity might be relevant in the metastatic process. The membranes of tumour cells with higher metastatic potential have a lower cholesterol/phospholipid ratio but greater unsaturated phospholipid content. This is partly supported by partition characteristics of metastatic variants in aqueous two-polymer phases. Steady-state fluorescence polarisation, which measures lipid order and the degree of rotational motion of lipids, does not suggest marked differences in bulk 'fluidity' of metastatic variants. Transbilayer fluidity differences have been described and these may be of some significance in the control of activity of membrane-associated enzymes and other membrane properties. The plasma membrane is a mosaic of domains possessing different degrees of microviscosity and this mosaicism may be relevant in the context of metastatic dissemination of tumours.

Hydrolysis of myelin basic protein in human myelin by terminal complement complexes.

The participation of terminal complement complexes (TCC) in demyelination has been shown in rodent cerebellar cultures. Since TCC modulates activities of various membrane-associated enzymes and increases the level of cellular Ca2+ we investigated whether TCC could activate Ca2+-dependent neutral proteases in myelin that would lead to hydrolysis of myelin basic protein (BP). Addition of antibody and C7-deficient serum plus C7 to sealed myelin vesicles of two to six bilayers caused significant BP hydrolysis compared to the hydrolysis caused by antibody and C7-deficient serum. Significant hydrolysis occurred at the stage of C5b6,7 assembly, which increased in magnitude at the C5b6-8 stage. C5b6-9 formation did not enhance the effect of C5b6-8. BP hydrolysis by C5b6,7 did not require Ca2+ whereas the effect of C5b6-8/C5b6-9 was, in part, Ca2+-dependent. We postulated that TCC formation in myelin membranes causes activation of myelin-associated neutral proteases with subsequent hydrolysis of BP as a consequence of complement peptide insertion and channel formation. Such processes may alter the structure of myelin and augment the action of other inflammatory cells and their products in demyelinating diseases that could ultimately lead to the loss of myelin.

24] Chemoattractant-induced membrane phenomena of phagocytes

This chapter discusses the several chemoattractant-induced membrane phenomena of phagocytes. Exposure of phagocytes to chemoattractants initiates a number of biological responses, including directed migration, secretion of hydrolytic enzymes, and production of cytotoxic oxygen species. These processes are activated by second messengers that are produced upon the interaction of chemoattractants with their receptors. The chapter also presents methods for studying chemoattractant-induced changes in phospholipid metabolism. Modification of the phospholipid composition of plasma membranes is a rapid response to many hormonal stimuli, including chemoattractants. Alterations of phospholipid metabolism can have pronounced effects on the activities of membrane-associated enzymes or produce second messengers act to regulate cell responses. The role of a guanine nucleotide regulatory (N) protein in phagocyte activation by chemoattractants is described. N proteins play pivotal roles in regulating the activity of adenylate cyclase and hence cyclic adenosine monophosphate (cAMP) levels in cells. Accumulating evidence suggests that N proteins may play similar roles in coupling phospholipase C activation to receptors that stimulate polyphosphoinositide breakdown. Methods described have been used predominantly for studying the responses of human polymorphonuclear leukocytes (PMNs) to the oligopeptide chemoattractant fMet-Leu-Phe but are also applicable to other chemoattractants.

Protein kinase C in pig thyroid cells: activation, translocation and endogenous substrate phosphorylating activity in response to phorbol esters

The phorbol ester, TPA, induced the intracellular redistribution of protein kinase C in intact thyroid cells; it caused within 5 min of incubation a 90% decrease of the cytosolic protein kinase C and an increase of the membrane-associated enzyme activity which appeared to be fully activated by TPA. TSH at concentrations which gave the maximal stimulation on various parameters of iodine metabolism induced the translocation of only 10–15% of protein kinase C from the cytosol to the membrane fraction. TPA induced a 2-fold increase in the incorporation of [ 32P]phosphate into cellular proteins and selectively activated the phosphorylation of two molecular species: a 180000 Da protein and to a lesser extent a 170000 Da protein in dispersed pig thyroid cells prelabeled with [ 32P]orthophosphate. The effect of TPA was maximum after 5 min of incubation and was concentration-dependent between 1 nM and 1 μM. The two phosphorylated substrates were only found in the cytosolic fraction. The TPA-induced phosphorylation of the 180000 Da protein was observed in thyroid cells in suspension, in thyroid cell monolayers and follicle-like reassociated cells. In these three experimental situations, the 180000 Da protein was not phosphorylated in response to TSH. Incubation of thyroid cell cytosolic fraction in the presence of [ 32P]ATP with calcium and phospholipid led to the phosphorylation of few proteins among which a 180000 Da component. These proteins were not phosphorylated in the cytosol of TPA-treated cells, a finding in agreement with the translocation of protein kinase C. These results indicate that (1) the activation-translocation of thyroid protein kinase C induced by TPA is associated with the phosphorylation of selective substrates, and (2) TSH, even at high concentration, failed to exert the same action as TPA on protein kinase C in pig thyroid cells.

Glial cells influence membrane-associated enzyme activity at the blood-brain barrier

Glial cells have been shown to influence several cerebral endothelial cell properties in vitro. A situation similar to the endothelial-astrocyte relationship existing at the blood-brain barrier (BBB) can be produced by growing cultured cerebral endothelium on one side of a filter and glial cells on the other in an enclosed double chamber. In this setting membrane-associated reaction product on the cerebral endothelial cell for both Na +, K +-ATPase and non-specific alkaline phosphatase was markedly increased when the endothelial cells were co-cultured with glial cells. In addition, the distribution of reaction product on the cerebral endothelial cell membrane was similar to the reported in vivo. These observations support a glial influence on enzyme activity at the BBB.

Changes of Some Membrane-Associated Enzyme Activities Degradation of Membrane Phospholipids in Cucumber Roots Due to Ca&lt;sup&gt;2+&lt;/sup&gt; Starvation

Changes of Some Membrane-Associated Enzyme Activities Degradation of Membrane Phospholipids in Cucumber Roots Due to Ca&lt;sup&gt;2+&lt;/sup&gt; Starvation

Subcellular membrane properties in vascular and non-vascular smooth muscles of Dahl hypertensive rats.

Subcellular membrane fractions were isolated from mesenteric arteries and vas deferens of salt-resistant and salt-sensitive Dahl rats on low-salt (0.4% NaCl) high-salt (8.0% NaCl) diets. Only the salt-sensitive Dahl rats on the high-salt diet developed sustained high blood pressure (BP) after 5 weeks of the high-salt diet. Protein contents, membrane associated enzyme activities, calcium ion (Ca2+) binding and ATP-dependent CA2+ transport were compared in fractions isolated from all four groups. No obvious changes were observed, except for minor enhancement in magnesium ion (Mg2+)- and CA2+ ATPase activities of mesenteric arterial membranes isolated from salt-sensitive Dahl rats on high-salt diet compared to those from other groups of rats. The membrane fractions from vas deferens of salt-sensitive Dahl rats on the high-salt diet, on the other hand, showed decreased ATP-dependent Ca2+ transport compared to those from salt-sensitive Dahl rats on the low-salt diet. No difference was observed in membrane fractions isolated from salt-resistant Dahl rats on high-salt diet compared to those on low-salt diet. The significance of these observations are discussed in relation to the findings previously obtained from corresponding smooth muscle tissues of spontaneous hypertensive rats (SHR).

Metabolic transformation of tritium-labeled pheromone by tissues ofHeliothis virescens moths

Unsaturated aliphatic pheromones ofH. VIRESCENS were prepared at high specific activity ((3)H, 58 Ci/mmol) and were employed to study tissue specificity of acetate esterase, alcohol oxidase, and aldehyde dehydrogenase in male and femaleHeliothis virescens. Thus, [9,10-(3)H2]Z9-14:Ac was synthesized by partial tritiation of the corresponding alkyne and was converted to the labeledZ9-14∶OH andZ9-14∶Al for metabolic studies. Soluble and membrane-associated enzyme activities were determined by radio-TLC assays. Esterase activity is highest in legs of both sexes, but also occurs in antennal and glandular tissues. Oxidase activity requires O2 and is highest in female pheromone gland tissues, but it is also high in the male hairpencils. Aldehyde dehydrogenase activity was uniformly high in all tissues, but highest in antennal tissues of both males and females.