Analogues of [3H] chloramphenicol for photoaffinity labeling.

The synthesis of [3H]chloramphenicol and its erythro-diastereoisomer with specific activities of 1.25 Ci/mmol, and the further transformation of the [3H]chloramphenicol to a series of azido and diazo-substituted derivatives are described. The antibiotic activity of the compounds was considered insufficient for their use as photoaffinity labels.     

Composition and labeling by [3H]glycerol and [3H]serine of phospholipids of the chicken retina and optic tectum

The content and fatty acid composition of phospholipids and the in vivo labeling of lipids by [3H]glycerol and [3H]serine was studied in the retina and the optic tectum of young chickens. The tectum had a higher content of phospholipids and a significantly lower ratio of choline (CGP) to ethanolamine (EGP) glycerophospholipids than the retina. Lipids of the chicken optic system were characterized by a high proportion of polyenoic fatty acids of the n-6 series compared to other species. Intravitreally injected [3H]glycerol was incorporated into all glycerol-containing lipids of the retina, especially in CGP and EGP. Most of the label from [3H]serine was found in serine glycerophospholipids (SGP). The time-dependent distribution of both precursors among retinal lipids was consistent with de novo synthesis as well as metabolic interconversions of lipids. Thus, [3H] from serine also appeared in EGP and CGP, indicating the presence and activity of SGP decarboxylase and EGP-n-methyl transferase. Lipids labeled with both precursors in retina were subsequently found in the tectum, via axoplasmic transport. Even though different lipid classes were labelled by each precursor the proportion of lipids transported to the tectum was similar in both cases (about 1% of the label present in retina).     

Modulation of specific [3H]phenytoin binding by benzodiazepines

We have shown that diazepam (ED₅₀ 2.4 M), flunitrazepam (ED₅₀ 10.2 M) and Ro5-4864 (ED₅₀ 5 M) are able to enhance both total and specific [³H]phenytoin binding. Picrotoxin (IC₅₀ 1.43 M) and chloride, either NaCl or KCl (IC₅₀ 42.4 M) inhibit both the increase in total and specific binding of [³H]phenytoin, Ro 15-1788 does not. The optimum time for this enhancement was 3–4 hours. While the ED₅₀'s for the benzodiazepines are high their order of potency suggests that an involvement of both the peripheral type benzodiazepine receptor and the GABA-chloride ionophore complex is likely. Clonazepam (IC₅₀ 23 M), oxazepam (IC₅₀ 12 M) chlordiazepoxide (IC₅₀ 35 M) and Ro8682-10, a convulsant benzodiazepine (IC₅₀ 16 M) all inhibit both total and specific [³H]phenytoin binding. These effects were not blocked by chloride ions, picrotoxin or Ro 15-1788, and reached equilibrium within 45 minutes. This order of potency also parallels that for the peripheral benzodiazepine receptor in rat brain. These data suggest the presence of a micromolar benzodiazepine receptor site which may play a role in the control of CNS excitability. Nitrazepam, medazepam, bromazepam and the tetralobenzodiazepines U38335, U42794, U43434, and U37834 had no effect on total or specific [³H]phenytoin binding nor on the actions of the other benzodiazepines described in concentrations up to 50 M.     

Muscarinic antagonist binding site heterogeneity as evidenced by autoradiography after direct labeling with [3H]-QNB and [3H]-pirenzepine

Saturable, high affinity binding of tritiated pirenzepine [( 3H]-PZ) was obtained in slide mounted tissue sections prior to performing autoradiographic localization of these binding sites. The binding in tissue sections of rostral rat forebrain gave a KD of 18nM and a Bmax of 51 fmoles/mg tissue. These binding characteristics are similar to those previously obtained in homogenate membrane preparations and indicate the binding is taking place in a similar manner. The distribution of the binding sites labeled with [3H]-PZ represented a subpopulation of those which could be labeled with tritiated quinuclidinyl benzilate [( 3H]-QNB). Thus, [3H]-PZ and [3H]-QNB both label regions of the cerebral cortex, hippocampus, striatum and dorsal horn of the spinal cord, while sites in the cerebellum, nucleus tractus solitarius, facial nucleus and ventral horn of the spinal cord are labeled with [3H]-QNB and not by [3H]-PZ. These observations indicate separate regions of the brain where antagonists bind to subtypes of muscarinic receptors.     

An improved method for labeling carboxyatractyloside by [3H]KBH4

Carboxyatractyloside was labeled with [3H]KBH4 after oxidation of the primary alcohol of the glucose disulfate moiety by dicyclohexylcarbodiimide and P2O5 under anhydrous conditions in a dimethylsulfoxide medium. The 3H-labeled product was purified by DE 52 column chromatography followed by Cellulofine GCL 25 column chromatography. The final 3H-labeled product gave a single spot on a thin-layer chromatogram, and its Rf value was the same as that of authentic carboxyatractyloside. The biological activities (such as inhibition of state 3 respiration and binding to the adenine nucleotide carrier) were also comparable with those of authentic carboxyatractyloside.     

Procaine isothiocyanate: An irreversible inhibitor of the specific binding of [3H]batrachotoxinin-A benzoate to sodium channels

[³H]Batrachotoxinin-A benzoate ([³H]BTX-B) binds with high affinity to sites on voltage sensitive sodium channels in synaptoneurosomes from guinea pig cerebral cortex. Local anesthetics competitively antagonize the binding of [³H]BTX-B. An irreversible local anesthetic, procaine isothiocyanate (PRIT) and a tritiated derivative ([³H]PRIT) have been prepared. PRIT inhibits the binding of [³H]BTX-B in a noncompetitive, irreversible manner (apparent K_i=13 M) whereas the parent compound, procaine, inhibits in a competitive, reversible manner (K_i=40 M). The dissociation rate of [³H]BTX-B from sites on the sodium channel is greatly accelerated in a concentration dependent manner in the presence of PRIT. A 50% increase in the dissociation rate of [³H]BTX-B is achieved in the presence of 0.98 M PRIT. [³H]PRIT binds irreversibly to three proteins in synaptoneurosomes with apparent molecular weights of 20, 42, and 68 kDa. Protection studies with procaine and other local anesthetics suggest that only the 68 kDa species was related to local anesthetic binding.     

Affinity labeling of yeast mitochondrial adenosine triphosphatase by reduction with [3H]borohydride.

Oligomycin-sensitive adenosine triphosphatase (ATPase) has been purified in large yields from yeast mitochondria by a procedure employing Sepharose 6B chromatography. The nature of the oligomycin binding site in this purified preparation has been studied by an affinity labeling technique in which oligomycin binding to the ATPase complex was followed by reduction of the complex with sodium [³H]borohydride. A major incorporation of label into protein with a molecular weight near 8000 was noted. This incorporation is dependent on the presence of oligomycin, is blocked by dicyclohexylcar-bodiimide, and is altered by mutations conferring oligomycin resistance to the ATPase. The evidence suggests that the low molecular weight proteolipid component of the ATPase complex is the site of oligomycin binding.     

Hepatic alpha-adrenergic receptors. Identification and subcellular localization using [3H]dihydroergocryptine.

Recently, several workers have shown that adrenergic control of hepatic carbohydrate metabolism has the characteristics of an alpha-receptor-mediated process. Using the rat liver membrane preparation of Neville (Neville, D. (1968) Biochim. Biophys. Acta 154, 540-552), alpha-adrenergic receptors have been identified using the ligand [3H]dihydroergocryptine. The receptors are saturable and of high affinity. Scatchard analysis yields a KD of 1.8 nM with 1.7 +/- 0.55 pmol of sites/mg of protein. Competition of dihydroergocryptine binding with various pharmacologic agents yields the typical (alpha-adrenergic potency series: (-)-epinephrine greater than (-)-norepinephrine greater than (-)-isoproterenol. (-)-Isomers are more potent than (+)-isomers. The alpha-blocker phentolamine is 3.4 orders of magnitude more potent than the beta-blocker propranolol. To determine subcellular localization of alpha-adrenergic receptors, livers were fractionated into a crude homogenate, a 1500 X g pellet, and the purified membrane preparation used previously for binding. Specific dihydroergocryptine binding, ouabain-inhibitable (Na,K)-ATPase, and F--stimulated adenylate cyclase activities, were followed in these fractions. Specific binding was enriched, relative to that in the crude homogenate, 2.88-fold in the pellet and 6.28-fold in the membranes. Similarly, (Na,K)-ATPase acticity was enriched 2.6-fold in the pellet and 7.1-fold in the membranes while adenylate cyclase activity was enriched 2.9-fold in the pellet and 3.5-fold in the membranes. It is concluded that hepatic alpha-adrenergic receptors are likely concentrated in the plasma membranes.     

Oestrogen and nuclear binding sites. Determination of specific sites by [3H]oestradiol exchange

A method was developed for the determination of the number of specific oestradiol-binding sites in the nuclear fraction of oestrogen-sensitive tissues. The method is based on the exchange of [(3)H]oestradiol with non-labelled oestradiol that is bound to nuclear binding sites. The number of specific nuclear binding sites after the injection of 2.5mug of oestradiol, an amount sufficient to saturate all binding sites, is 1.6-1.7pmol per immature uterus. The number of sites occupied after an injection of physiological amounts of oestradiol (0.1mug) was 0.46pmol. The injection of oestradiol results in an increased number of nuclear binding sites in uterus and vagina, but has no effect on kidney or muscle. Injections of testosterone or progesterone failed to increase the number of uterine nuclear binding sites. This method permits an evaluation of the number of oestradiol-binding sites in the nuclear fraction of various tissues as a function of either endogenous oestradiol or non-labelled oestradiol administered by injection.     

Selective labeling of alpha-noradrenergic receptors in rat brain with [3H]dihydroergokryptine.

Using concentrations of [³H] dihydroergokryptine between 0.1 and 5 nM, saturable binding can be demonstrated in rat cerebral cortical membranes with a dissociation constant (K_D) of about 0.8 nM. α-Noradrenergic agonists and antagonists compete for the sites labeled by these low concentrations of [³H] dihydroergokryptine with relative potencies characteristics of classical α-noradrenergic receptors. The very low potency of serotonin in competing for these binding sites indicates that, in contrast to findings with higher concentrations of [³H] DHE, low concentrations do not label serotonin receptors. Moreover, the low potency of dopamine in competing for [³H] dihydroergokryptine binding in both striatal and cortical membranes indicates that no detectable portion of binding is associated with postsynaptic dopamine receptors.     

Perturbation of DNA replication and cell cycle progression by commonly used [3H]thymidine labeling protocols.

The effect of tritiated thymidine incorporation on DNA replication was studied in Chinese hamster ovary cells. Rapidly eluting (small) DNA from cells labeled with 2 microCi of [3H]thymidine per ml (200 microCi/mmol) for 60 min matured to a large nonelutable size within approximately 2 to 4 h, as measured by the alkaline elution technique. However, DNA from cells exposed to 10 microCi of [3H]thymidine per ml (66 microCi/mmol) was more rapidly eluting initially and did not mature to a nonelutable size during subsequent incubation. Semiconservative DNA replication measured by cesium chloride gradient analysis of bromodeoxyuridine-substituted DNA was also found to be affected by the final specific activity of the [3H]thymidine used in the labeling protocol. Dramatic cell cycle perturbations accompanied these effects on DNA replication, suggesting that labeling protocols commonly used to study DNA metabolism produce aberrant DNA replication and subsequent cell cycle perturbations.     

Trypanosoma cruzi: incorporation of [3H]-palmitic acid and [3H]-galactose into components shed by trypomastigotes.

Trypomastigotes were metabolically labeled with [3H]-palmitic acid or [3H]-galactose and labeled components were detected in the culture medium. Thin layer chromatography of the shed material showed several lipids in the [3H]-palmitic acid labeled sample while the sugar was mainly incorporated into macromolecules. The material incorporated with the lipidic precursor was fractionated by DEAE-Sephadex (acetate form) and the amount of radioactivity was ten times higher in the acidic lipids than in the neutral lipids. When acidic lipids were further separated by Unisil, 73% of the radioactivity was recovered in the less polar fraction. Different patterns were obtained on comparison of the shed components with the lipids remaining in the parasite.     

Photoaffinity labeling of [3H]flunitrazepam- and [3H]Ro15-4513-bound pellets in rat cerebral cortex and cerebellum

Irreversible incorporation of [3H]flunitrazepam and [3H]Ro15-4513 into GABA/benzodiazepine receptor subunits was studied by UV irradiation using ligand-bound membrane pellets from rat cerebral cortical and cerebellar synaptic membranes. Specific incorporation for [3H]flunitrazepam was greater in the pellet than in the suspension. The incorporation was identical for [3H]Ro15-4513 in both pellet and suspension. With the ligand-bound pellets, 50% of the available binding sites were photolabeled by both ligands in cortex and cerebellum. SDS polyacrylamide gel electrophoresis and fluorography of [3H]flunitrazepam photo-labeled receptor revealed the same number of major sites in both brain regions. In contrast, [3H]Ro15-4513 appears to label fewer sites in cortex and cerebellum. Photoaffinity labeling with [3H]flunitrazepam in ligand-bound membrane pellet provides a more selective and reliable method for studying the subunit structure of GABA/benzodiazepine receptor complex.     

[3H]Tyramine binding: A comparison with neuronal [3H]-dopamine uptake and [3H]mazindol binding processes

[³H]Spiperone ([³H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [³H]tyramine ([³H]PTA), thus indicating that [³H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [³H]PTA binding and [³H]DA uptake processes were markedly decreased, with no effect on [³H]mazindol ([³H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [³H]PTA binding, did not affect the [³H]MAZ binding process. This further supported the suggestion that while [³H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [³H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.Special issue dedicated to Dr. Paola S. Timiras     

Direct photoaffinity labeling of the primary region of the ouabain binding site of (Na+ + K+)-ATPase with [3H]ouabain, [3H]digitoxin and [3H]digitoxigenin.

The tritiated cardiotonic steroids, ouabain, digitoxin, and digitoxigenin are shown to photolabel the large polypeptide but not the glycoprotein or proteolipid component of the (Na+ + K+)-ATPase when they are bound to the inhibitory site and exposed to light of 220 or 254 nm. The extent of photolabeling is low, less than 1%, and is limited by photocross-linking of the enzyme. The mechanism of photoincorporation does not appear to be either photolysis of the lactone ring in ouabain or photolysis of tryptophan or tyrosine residues in the polypeptide.     

Different specific binding sites of [3H]glycine and [3H]strychnine in synaptosomal membranes isolated from frog retina

Synaptosomal fractions were isolated from frog retina: a fraction enriched in photoreceptor terminals (P1) and a second one (P2) containing interneurons terminals. We compared the binding of [³H]glycine and [³H]strychine to membranes of these synaptosomes. The binding of both radioactive ligands was saturable and Na⁺-independent. [³H]Glycine bound to a single site in P1 and P2 synaptosomal fractions, with K_D=12 and 82 nM and B_Max=3.1 and 3.06 pmol/mg protein respectively. [³H]Strychnine bound to two sites in each one of the synaptosomal fractions. For P1 K_D values were 3.9 and 18.7 nM, and B_Max values were 1.1 and 7.1 pmol/mg protein, respecitively. Membranes from the P2 synaptosomal fraction showed K_D's of 0.6 and 48 nM and B_Max's of 0.4 and 4.5 pmol/mg. Specific [³H]glycine binding was displaced by -alanine, l-serine, d-serine and HA966, but not by strychnine 7-chlorokynurenic or 5,7-dichloro-kynurenic acids. Specific [³H]strychnine, binding was partially displaced by glycine and related aminoacids and totally displaced only by 2-NH₂-strychnine. Our results indicate the presence of high affinity binding sites for glycine and strychnine in frog retinal synaptosomal membranes. The pharmacological binding pattern indicates the presence of the strychnine sensitive glycine receptor as well as other sites. These might not include the NMDA receptor-associated glycine site.     

3H]epinephrine and [3H]norepinephrine binding to alpha-noradrenergic.

(±)-[³H]Epinephrine and (?)-[³H]norepinephrine bind saturably to calf cerebral cortex membranes under appropriate incubation conditions in a fashion indicating that they label α-noradrenergic receptors. Binding of the two [³H]catecholamines is saturable with dissociation constants of 20–30 nM. Binding is stereoselective with (?)-norepinephrine displaying about twenty times greater affinity than (+)-norepinephrine. The relative potencies of catecholamines in competing for these binding sites parallels their relative pharmacologic effects at α-noradrenergic receptors in numerous tissues. Thus, (?)-epinephrine is 2–3 times more potent than (?)-norepinephrine and 500 times more potent than (?)-isoproterenol. Binding is inhibited by low concentrations of the α-antagonists phentolamine and phenoxybenzamine but not by the β-antagonist propranolol.