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PubMed1996

Melanocortin probes for the melanoma MC1 receptor: synthesis, receptor binding and biological activity.

The fate of alpha-melanocyte-stimulating hormone (alpha-MSH) subsequent to binding to the melanoma melanocortin MC1 receptor is of interest with regard to its potential use in targeting cytotoxic drugs or imaging to melanoma. Tools such as iodinated, photoaffinity-labelled, biotinylated and fluorescent melanocortins are required to study the fate of the ligand during its interaction with the receptor. In this study, a series of probes for the receptor based on the potent analogue, [Nle4,Dphe7]alpha-MSH, have been developed and tested for their potential usefulness. All probes contain the core melanocortin motif His-Phe-Arg-Trp. They bind the receptor readily and appear to have similar intrinsic efficacies to the endogenous peptide, so that biological activity is regulated by their receptor binding affinity. Functional photoaffinity-labelled, biotinylated and fluorescent probes are described. The biotinylated probe binds the receptor when coupled to streptavidin, although with reduced affinity.

Animals↗

PubMed1993

Evidence that lipopolysaccharide and pertussis toxin bind to different domains on the same p73 receptor on murine splenocytes.

In previous studies, we used a photoactivable, radioiodinated lipopolysaccharide (LPS) derivative to define and characterize a specific bacterial endotoxic LPS-binding protein (p73) on mammalian lymphoreticular cells, including B and T lymphocytes and macrophages. More recently, using the same methodology, we characterized a specific interaction of LPS with the S2 subunit of Bordetella pertussis pertussis toxin (PT) in the fluid phase (M.-G. Lei and D. C. Morrison, J. Biol. Chem., 268:1488-1493, 1993). Furthermore, we showed that lysozyme (LZM) but not polymyxin B can compete with PT for binding to LPS in the fluid phase, a result suggesting that these two molecules compete for the same binding site on LPS. In this report, we demonstrate that the binding of PT to murine splenocytes (cell-bound PT) reduces the ability of the LPS photo-cross-linking probe to bind to the p73 receptor. The reduction can also be demonstrated with the PT B oligomer, a result indicating that the observed reduction of LPS binding to the p73 receptor by PT is A-protomer (S1-subunit) independent. More importantly, our studies document that cell-bound PT can be radiolabelled by the LPS probe, coincident with the observed reduction in p73 photoaffinity labelling. The preferential interaction of LPS with the PT S2 subunit in the fluid phase was, however, not observed with cell-bound PT. The reduction in radiolabelling of the p73 receptor by the LPS probe and in radiolabelling of cell-bound PT was shown to be concentration dependent. The data presented here document, however, that LZM does not reduce the ability of the LPS probe to bind to the p73 receptor on mouse splenocytes, nor does the presence of LZM bound to LPS influence the observed reduction in photoaffinity labelling of p73 by the LPS probe or radiolabelling of cell-bound PT by the LPS probe. Collectively, these results support the concept that the ability of LPS to interact with PT in the fluid phase is not responsible for the ability of cell-bound PT to influence the binding of the LPS probe to the p73 receptor. Thus, it is suggested that PT and LPS bind to different sites on the p73 molecule and that this same p73 protein may recognize both LPS and PT.

Animals↗

PubMed1976

Production of frameshift mutations in Salmonella by a light sensitive azide analog of ethidium.

Frameshift mutations have been produced in specific repair-negative Salmonella tester strains by photoaffinity labeling technique using ethidium azide. Reversions requiring a +1 addition or a -2 deletion were specially sensitive. Mutagenesis was reduced by the simultaneous addition of non-mutagenic ethidium bromide, and was prevented by photolysis of the azide prior to culture addition. Identical tester strains active in DNA excision repaire were not mutagenized by the azide. These results are consistent with the interpretation that photolysis of the bound ethidium analog converts the drug from its noncovalent mode of binding (presumably intercalation) to a covalent complex with consequent production of frameshift mutations. Such photoaffinity labeling by drugs which bind to DNA not only confirms the importance of covalent drug attachment for frameshift mutagenesis, but also provides powerful techniques for studying the molecular deatils of a variety of genetic mechanisms.

Azides↗

PubMed1990

Photolabelling of the prostaglandin E2 receptor in cardiac sarcolemmal vesicles.

A [3H]azidophenacyl ester of PGE2 ([3H]azido-PGE2) was synthesized and used to photoaffinity label the protein component of the high affinity PGE2 binding site in cardiac sarcolemma membrane. Photolysis of the isolated cardiac sarcolemmal vesicles in the presence of [3H]azido-PGE2 resulted in the covalent labelling of a protein component that migrated on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of 100,000. Incorporation of the [3H]azido-PGE2 did not occur in the absence of photolysis. The photolabelling of the 100-kDa protein by [3H]azido-PGE2 was inhibited by excess unlabelled PGE2 and azido-PGE2. Specific binding of [3H]azido-PGE2 was displaced by excess unlabelled PGE2 or azido-PGE2, but not PGF2 alpha, 6-keto-PGF1 alpha or PGD2. These results indicate that the 100-kDa photoaffinity labelled [3H]azido-PGE2 binding protein contains the binding site for PGE2 in isolated cardiac sarcolemma membranes.

Affinity Labels↗

PubMed1990

Location of transported auxin in etiolated maize shoots using 5-azidoindole-3-acetic Acid.

A study was undertaken using the photoaffinity labeling agent, tritiated 5-azidoindole-3-acetic acid ([(3)H],5-N(3)IAA), to identify cells in the etiolated maize (Zea mays L.) shoot which transport auxin. Transport of [(3)H],5-N(3)IAA was shown to be polar, inhibited by 2,3,5-triiodobenzoic acid (TIBA) and essentially freely mobile. There was no detectable radiodecomposition of [(3)H],5-N(3)IAA within tissue kept in darkness for 4 hours. Shoot tissue which had taken up [(3)H],5-N(3)IAA was irradiated with ultraviolet light to covalently fix the photoaffinity labeling agent within cells that contained it at the time of photolysis. Subsequent microautoradiography showed that all cells contained radioactivity; however, the amount of radioactivity varied among different cell types. Epidermal cells contained the most radioactivity per area, approximately twofold more than other cells. Parenchyma cells in the mature stelar region contained the next largest amount and cortical cells, sieve tube cells, tracheary cells, and all cells in the leaf base contained the least amount of the radioactive label. Two observations suggest that the auxin within the epidermal cells is transported in a polar manner: (a) the amount of auxin in the epidermal cells is greatly reduced in the presence of TIBA, and (b) auxin accumulates on the apical side of a wound in the epidermis and is absent on the basal side. While these results indicate that auxin in the epidermis is polarly transported, this tissue cannot be the only pathway since the epidermis is only a small fraction of the shoot volume. The greater than twofold difference between the concentration of auxin in the epidermal and subtending cells demonstrates that physiological differences in the concentration of auxin can occur between adjacent cells.

Journal Article↗

PubMed2001

Characterization of the dizocilpine binding site on the nicotinic acetylcholine receptor.

Although the dissociative anesthetic dizocilpine [(+)-MK-801] inhibits nicotinic acetylcholine receptor (AChR) function in a noncompetitive manner, the location of the dizocilpine binding site(s) has yet to be clearly established. Thus, to characterize the binding site for dizocilpine on the AChR we examined 1) the dissociation constant (K(d)) and stoichiometry of [(3)H]dizocilpine binding; 2) the displacement of dizocilpine radioligand binding by noncompetitive inhibitors (NCIs) and conversely dizocilpine displacement of fluorescent and radiolabeled NCIs from their respective high-affinity binding sites on the AChR; and 3) photoaffinity labeling of the AChR using (125)I-dizocilpine. The results establish that one high-affinity (K(d) = 4.8 microM) and several (3-6) low-affinity (K(d) = approximately 140 microM) binding sites exist for dizocilpine on the desensitized and resting AChR, respectively. The binding of the fluorescent NCIs ethidium, quinacrine, and crystal violet as well as [(3)H]thienylcyclohexylpiperidine was inhibited by dizocilpine on desensitized AChRs. However, Schild-type analyses indicate that only the inhibition of quinacrine in the desensitized state seems to be mediated by a mutually exclusive action. Photoaffinity labeling of the AChR by (125)I-dizocilpine was primarily restricted to the alpha1 subunit and subsequent mapping revealed that the principal sites of labeling are localized to the M4 (approximately 70%) and M1 (30%) transmembrane domains. Collectively, the data indicate that the high-affinity dizocilpine binding site is not located in the lumen of the ion channel but probably near the quinacrine binding locus at a nonluminal domain in the AChR desensitized state.

Animals↗

PubMed1995

Synthesis and applicability of a photolabile 7,7-azi analogue of 3-sulfated taurine-conjugated bile salts.

For the identification of proteins involved in hepatobiliary transport, the photolabile derivative 7,7-ASLCT ((7,7-azi-3 alpha-sulfato-5 beta-cholan-24-oyl)-2'-aminoethanesulfonate) was synthesized. 7,7-ASLCT is taken up into liver and excreted into bile completely unmetabolized at a rate between the excretion rate of SLCT ((3 alpha-sulfato-5 beta-cholan-24-oyl)-2'- aminoethanesulfonate) and SCCT ((7 alpha-hydroxy-3 alpha-sulfato-5 beta- cholan-24-oyl)-2'-aminoethanesulfonate). The dependency of flux rate of uptake into freshly isolated hepatocytes on the concentration of 7,7-ASLCT in presence of Na+ (143 mM) and with Na+ depletion (1 mM) is best described by the assumption of two simple transport systems, the kinetic parameters of which are similar to those of SLCT. As studied in the presence of Na+, 7,7-ASLCT and SLCT exhibit a clearly competitive cross-inhibition of uptake with inhibition constants that are similar to the corresponding half-saturation constants. Photoaffinity labeling of isolated hepatocytes using 7,7-ASLCT (400 microM) resulted in the irreversible inhibition of the uptake of 7,7-ASLCT and SLCT to similar extents, confirming the kinetic data that 7,7-ASLCT is a true competing substrate for the uptake of SLCT. Because in intact rat liver 7,7-ASLCT and SLCT mutually inhibit their biliary excretion, the photolabile derivative shares with SLCT the same pathways in uptake and in excretion. Thus, 7,7-ASLCT should be appropriate for the study of hepatobiliary transport of sulfated and taurine-conjugated bile salts by photoaffinity labeling.

Affinity Labels↗

PubMed1990

Pseudomonas aeruginosa exotoxin A: alterations of biological and biochemical properties resulting from mutation of glutamic acid 553 to aspartic acid.

Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) was identified earlier as a putative active-site residue by photoaffinity labeling with NAD. Here ETA-E553D, a cloned form of the toxin in which Glu-553 has been replaced by aspartic acid, was purified from Escherichia coli extracts and characterized. Cytotoxicity of the mutant toxin for mouse L-M cells was less than 1/400,000 that of the wild type. The mutation caused a 3200-fold reduction in NAD:elongation factor 2 ADP-ribosyltransferase activity, as estimated by assays with an active fragment derived from the toxin by digestion with thermolysin. NAD glycohydrolase activity was reduced somewhat less, by a factor of 50, and photoaffinity labeling with NAD by a factor of 2. We detected less than 2-fold change in the values of KM for NAD or elongation factor 2 and no change in KD for NAD, as determined by quenching of protein fluorescence. The drastic reduction of ADP-ribosyltransferase activity therefore results primarily from an effect of the mutation on kcat, implying that Glu-553 plays an important and possibly direct role in catalyzing this reaction. The effects of the E553D mutation are similar to those of the E148D mutation in diphtheria toxin, supporting the notion that these two Glu residues perform the same function in their respective toxins.

ADP Ribose Transferases↗

PubMed1995

Photochemical reactions of azidocoumarins.

Photochemical reactions of 6-azidocoumarin and 7-azido-4-methylcoumarin in the presence of secondary amines have been investigated for their potential applications in photoaffinity labeling. It was found that the singlet nitrene generated from 6-azidocoumarin isomerized to a dehydroazepine intermediate that reacted with an amine to yield two isomeric adducts. Photolysis of 7-azido-4-methylcoumarin, in contrast, gave a triplet nitrene that abstracted hydrogen atoms from secondary amine molecules to form 7-amino-4-methylcoumarin as the major product. The difference in the intersystem crossing rate between the two compounds originates from the azido position relative to the carbonyl group. Because of its ability to form a covalent linkage with a nucleophile, 6-azidocoumarin is deemed to have a greater potential as a photoaffinity label than 7-azido-4-methylcoumarin.

Affinity Labels↗

PubMed1993

Biphasic intracellular redistribution of alpha 1-adrenergic receptors in rat liver during sepsis.

Changes in the distribution of alpha 1-adrenergic receptors in two subcellular fractions, the plasma membrane and the light vesicle, of rat liver during sepsis were studied using [3H]prazosin binding and photoaffinity labeling with [125I]arylazidoprazosin in combination with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit two metabolically distinct phases: an initial hypermetabolic (hyperglycemic) phase (9 h after CLP; early sepsis) followed by a hypometabolic (hypoglycemic) phase (18 h after CLP; late sepsis). [3H]prazosin binding studies show that during early sepsis, the maximal binding capacity (Bmax) was increased by 35% in plasma membranes but was decreased by 28% in light vesicles; while during late sepsis, the Bmax was decreased by 30% in plasma membranes but was increased by 33% in light vesicles. The photoaffinity labeling studies revealed two major binding peptides with M(r) of 77,000 and 68,000 Da and one minor binding protein with M(r) of 39,000 Da. The total binding for the three labeled peptides during early sepsis was increased by 26% in plasma membranes but was decreased by 33% in light vesicles, while during late sepsis the total binding was decreased by 19% in plasma membranes but was increased by 35% in light vesicles. These data indicate that alpha 1-adrenergic receptors in the rat liver were externalized from light vesicles to plasma membranes during the hyperglycemic phase while they were internalized from surface membranes to intracellular sites during the hypoglycemic phase of sepsis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Benzodiazepine receptor subunits in avian brain.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of the photoaffinity-labeled benzodiazepine receptor in duck brain, two equally intensive bands of relative molecular masses of 53,000 and 54,000 were observed, thus being identical to the subunit pattern previously described for chicken brain. An attempt was made to characterize further the avian subunits. Comparison of the fluorographic subunit pattern in cerebellum and telencephalon revealed a pronounced quantitative heterogeneity. Addition of ethyl beta-carboline-3-carboxylate resulted in the selective inhibition of the 53,000 subunit. These results are compared with the mammalian subunit pattern in the corresponding brain regions. The findings are discussed in view of the apparent contradiction between the existence of regional heterogeneity when photoaffinity labeling is applied and its lack when monoclonal antibodies are used.

Affinity Labels↗

PubMed1999

[3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors.

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [3H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [3H]CGP 61594 labeled with high affinity (K(D) = 23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [3H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [3H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [3H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors.

Affinity Labels↗

PubMed1991

Developmental regulation of the distribution of rat brain insulin-insensitive (Glut 1) glucose transporter.

We examined the immunohistochemical localization of Glut 1 in sections of developing rat brain [gestational (G) days 18 and 20, postnatal (P) days 1, 10, 21, and greater than 40; term = approximately G21] and characterized the abundance of Glut 1 in isolated brain microvessels by Western blot analysis. Further quantitation of total glucose transporters by [125I]3-iodo-4-azido-phenethyl-amido-7-O-succinyldeacetyl-forskoli n photoaffinity labeling was performed. Glut 1 was prominently distributed in G18 and P1 brain vascular endothelial cells, with comparatively little immunoreactivity observed in brain parenchyma. Conversely, at P10, Glut 1 was prominent in brain parenchyma (undifferentiated cells) and less evident in the vascular endothelium. However, at P21, a resurgence of Glut 1 in vascular endothelial cells was observed, as was a sustained presence in parenchymal cells. On P greater than 40, a distribution in vascular endothelial cells and, to a minor extent, in parenchymal perivascular stellate cells was noted. Microvessel preparation Glut 1 (approximately 55-60 kDa) and [125I] 3-iodo-4-azido-phenethyl-amido-7-O-succinyldeacetyl-forskolin photoaffinity-labeled glucose transporters gradually increased from G18 through P10 to P greater than 40. This developmental increase in Glut 1 was also seen in whole homogenates (approximately 45-47 kDa), but not in crude brain membranes. Thus, isolated microvascular preparations indicated that Glut 1 levels constantly increased with maturation, but direct visualization of brain sections revealed that the localization in different cellular compartments changed with development. These alterations in cellular localization of glucose transporters, appear to occur in concert with the changing glucose metabolic needs of the brain, which, during the various stages of development, exhibits constantly changing phases of cellular growth and proliferation.

Aging↗

PubMed1989

Two progesterone receptors in the oviduct of the freshwater turtle Chrysemys picta: possible homology to mammalian and avian progesterone receptor systems.

Two progesterone receptors in the oviduct of the freshwater turtle Chrysemys picta: possible homology to mammalian and avian receptor systems. Here we report the characterization of two specific progesterone receptors in nuclear extracts of the turtle oviduct. The receptors differ in dissociation constants (2.8 nM vs 27 nM) which can be separated on DEAE-Sepharose, the former eluting at 0.08 M KCl and the latter at 0.20 M KCl. [3H]R5020 photoaffinity labeling SDS-PAGE revealed that the 2.8 nM moiety migrates with an apparent molecular weight of 80 +/- 5 kDa and the 27 nM moiety migrates with an apparent molecular weight of 120 +/- 5 kDa. These receptors are termed PR-A and PR-B due to their molecular mass and elution profiles. DNA-cellulose chromatographic studies show that both bind DNA-cellulose with the PR-A eluting at 0.09 M NaCl and PR-B eluting between 0.20-0.21 M NaCl. In reproductively inactive turtles (from the months of January and February) estradiol is undetectable, and PR-B is absent as determined by Scatchard analysis, [3H]R5020 photoaffinity labeling electrophoretic studies and DEAE-Sepharose and DNA-cellulose chromatography. In these animals PR-B can be replenished by estrogen treatment, suggesting a physiological role for both PR-A and PR-B and dependence of PR-B on estradiol.

Animals↗

PubMed2006

1-Benzyl-1,2,3,4-tetrahydroisoquinoline-6,7-diols as novel affinity and photoaffinity probes for beta-adrenoceptor subtypes.

Trimetoquinol (TMQ, 1) is a potent non-selective beta-adrenoceptor (beta-AR) agonist possessing a tetrahydroisoquinoline (THI) structure. The binding site for 1-trimethoxybenzyl group of 1, which distinguishes it from classical catecholamines, is unknown. Affinity and photoaffinity labeled compounds are good tools to determine the exact interaction between a ligand and a specific amino acid(s) in a receptor. In this study, we designed and synthesized a series of affinity 6, 12, 18, and photoaffinity 24, 29 labeled analogues of TMQ. All of these compounds were full agonists and demonstrated an equal or greater binding affinity and functional activity as compared to TMQ on beta1-, beta2-, and beta3-AR. Washout experiments on Chinese hamster ovary (CHO) cells expressing hu beta2-AR were helpful in identifying the isothiocyanate 18 and the azide 24 as very effective affinity and photoaffinity labels at this receptor subtype.

Adrenergic beta-Agonists↗

PubMed1996

Interaction of cytostatics and chemosensitizers with the dexniguldipine binding site on P-glycoprotein.

The interaction of cytostatics and chemosensitizers with the dexniguldipine binding site of P-glycoprotein was investigated in photoaffinity labeling experiments. A tritiated azidoderivative of the chemosensitizer dexniguldipine with dihydropyridine structure, [3H]B9209-005, was used to irreversibly label P-glycoprotein. The apparent affinity of cytostatics and chemosensitizers to this binding site was estimated from labeling experiments in the presence of increasing concentrations of compounds. From the cytostatics tested, the vinca alkaloids and taxol showed the highest affinity, anthracyclins possessed moderate affinity while methotrexate, ara C and camptothecin, cytostatics not involved in P-glycoprotein-mediated multidrug resistance, were almost inactive. The chemosensitizers GF 120918, cyclosporin A and SDZ PSC-833 inhibited photoincorporation with the highest potency. Steep dose-inhibition curves were obtained with the cyclic peptides and S9788, indicating that these compounds may bind allosterically to a separate binding site. Compounds with dihydropyridine structure with or without chemosensitizing potency were also tested and some structure-activity relationships could be derived from the data. Our data show that inhibition of photoaffinity labeling by [3H]B9209-005 is a valuable and reliable system for measuring the interaction with and potency of chemosensitizing compounds at P-glycoprotein. Furthermore, data obtained in this test system are well suited to investigate structure-activity relationships for chemosensitizers at P-glycoprotein. In addition cytostatics underlying P-glycoprotein-mediated multidrug resistance can be identified.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Regulatory interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton in human neutrophils.

The cytoskeleton and/or membrane skeleton has been implicated in the regulation of N-formyl peptide receptors. The coupling of these chemotactic receptors to the membrane skeleton was investigated in plasma membranes from unstimulated and desensitized human neutrophils using the photoreactive agonist N-formyl-met-leu-phe-lys-N epsilon-[125I]2(p-azidosalicylamido)ethyl-1,3'- dithiopropionate (fMLFK-[125I]ASD). When membranes of unstimulated cells were solubilized in Triton-X 100, a detergent that does not disrupt actin filaments, only 50% of the photoaffinity-labeled receptors were solubilized sedimenting in sucrose density gradients at a rate consistent with previous reports. The remainder were found in the pellet fraction along with the membrane skeletal actin. Solubilization of the membranes in the presence of p-chloromercuriphenylsulfonic acid, elevated concentrations of KCl, or deoxyribonuclease I released receptors in parallel with actin. When membranes from neutrophils, desensitized by incubation with fMLFK-[125I]ASD at 15 degrees C, were solubilized, nearly all receptors were recovered in the pellet fraction. Incubation of cells with the ligand at 4 degrees C inhibited desensitization partially and prevented the conversion of a significant fraction of receptors to the form associated with the membrane skeletal pellet. In these separations the photoaffinity-labeled receptors not sedimenting to the pellet cosedimented with actin. Approximately 25% of these receptors could be immunosedimented with antiactin antibodies suggesting that N-formyl peptide receptors may interact directly with actin. These results are consistent with a regulatory role for the interaction of chemotactic N-formyl peptide receptors with actin of the membrane skeleton.

Actin Cytoskeleton↗

PubMed1994

Cyclic AMP-reactive proteins in human saliva.

This study was conducted to compare cyclic AMP-reactive proteins (cARP), the secretory form of regulatory (R) subunits of cyclic AMP-dependent protein kinase (PKA), and cyclic nucleotide phosphodiesterase (PDE) activity in human whole saliva with that of parotid fluid. Additionally, experiments were done to determine whether secretory cARP is altered by environmental stimuli. Earlier work showed that R subunits are present in parotid fluid and in salivary glands of rats. No previous information is available about secretory PDE in saliva. Whole and parotid ductal saliva samples were collected by a non-invasive procedure from healthy volunteers. After photoaffinity labelling with [32P]-8-N3-cAMP, the R subunits were identified by autoradiography. Cyclic nucleotide PDE activity was measured as a function of the conversion of the cyclic nucleotide to the tritiated 5'-nucleotide. The results showed that R of the type II cAPK, RII (M(r) 50-54 kDa) and/or a slower-moving isoform (M(r) 54-56 kDa, RIIa) were present in all parotid saliva samples tested. Whole saliva was positive for RII in more than 95% of the samples tested (n = 62), but with 50-90% reduction in concentration compared to parotid fluid. Both female and male subjects exposed to controlled auditory (60-80 dB) stimuli responded by a two- to five-fold increase in photoaffinity labelling of cARP (salivary RII, RIIa and RIIfr). There was considerable individual variability, but in all cases the differences in the results were significant (p < 0.05, n = 20). Whole saliva showed measurable PDE activity in fresh or frozen samples, whereas no PDE activity was detected in parotid fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗