Sedimentary 12-n-Propylcholestanes, Molecular Fossils Diagnostic of Marine Algae.

Certain C(30)-steranes have been used for identifying sedimentary rocks and crude oils derived from organic matter deposited in marine environments. Analysis of a C(30)-sterane from Prudhoe Bay oil indicates that these C(30)-steranes are 24-n-propylcholestanes that apparently are derived from precursor sterols 24-n-propylidene-cholesterols and 24-n-propylcholesterol. These widely occurring sterols are biochemically synthesized in modern oceans by members of an order (Sarcinochrysidales) of chrysophyte algae. These data thus imply that C(30)-sterane biomarkers in sedimentary rocks and crude oils have a marine origin. Screening of a few organic-rich sedimentary rocks and oils from throughout the Phanerozoic suggests that these C(30)-steranes first appeared and, therefore, their source algae evolved between Early Ordovician and Devonian.

Prostaglandin photoaffinity probes: synthesis and biological activity of azide-substituted 16-phenoxy- and 17-phenyl-PGF2 alpha prostaglandins.

The development of a prostaglandin PGF2 alpha photoaffinity probe led to the synthesis and biological evaluation of azide-substituted 17-phenyl-18,19,20-trinorprostaglandin F2 alpha and 16-phenoxy-17,18,19,20-tetranorprostaglandin F2 alpha derivatives. Two approaches for the preparation of iodinated versions of these prostaglandins were evaluated: (1) iodination of a phenyl azide bearing an activating hydroxyl group and (2) iodination of an aniline precursor to the phenyl azide group and subsequent conversion of the aniline to the phenyl azide. In the first approach, 17-(4-azido-2-hydroxyphenyl)-18,19,20-trinorprostaglandin F2 alpha, 16-(5-azido-3-hydroxyphenoxy)-17,18,19,20-tetranorprostaglandin F2 alpha, and 16-(4-azido-2-hydroxyphenoxy)-17,18,19,20-tetranorprostaglandin F2 alpha were prepared by using the Corey synthesis, but were biologically inactive presumably as a result of the hydrophilic phenolic hydroxyl group. In the second approach, the iodination of a 17-(4-aminophenyl)-18,19,20-trinorprostaglandin F2 alpha derivative delivered 17-(4-azido-3-iodophenyl)-18,19,20-trinorprostaglandin F2 alpha, which exhibited competitive binding with natural [3H]PGF2 alpha to ovine luteal cells and to plasma membranes of bovine corpora lutea. [125I]-17-(4-Azido-3-iodophenyl)-18,19,20-trinorprostaglandin F2 alpha was utilized in a preliminary photoaffinity cross-linking experiment.

Structural requirements for prostaglandin analog interaction with the ovine corpus luteum prostaglandin F2 alpha receptor. Implications for development of a photoaffinity probe.

The capacity of structurally modified analogs of prostaglandin F2 alpha (PGF2 alpha) to inhibit binding of [3H]PGF2 alpha to receptors on ovine luteal cells was evaluated by radioreceptor assay using dispersed, viable, ovine luteal cells. Binding assays were conducted at pH 5.75, since binding to both high (Kd 17.4 +/- 2.3 nM) and low (Kd 409 +/- 166 nM) affinity sites was enhanced markedly at reduced pH. The capability to compete with [3H]PGF2 alpha for binding was evaluated for different prostaglandin analogs having modifications in the C-8 "upper" side-chain, in the cyclopentane ring, or in the C-12 "lower" side-chain. Prostaglandin J2 was a surprisingly potent competitor for binding to the PGF2 alpha receptor. Several phenyl-substituted analogs exhibited receptor-binding potency greater than or equal to native PGF2 alpha, while most other analogs had reduced capacity to compete with native PGF2 alpha for binding. Several 17-azidophenol PGF2 alpha analogs were synthesized and tested, but analogs having hydroxyl groups on the aryl ring had low affinity for receptors. However, 17-(4-azidophenyl)-18,19,20-trinor-PGF2 alpha as well as 17-(3-iodo-4-azidophenyl)-18,19,20-trinor-PGF2 alpha exhibited binding affinities that were approximately 10% of native PGF2 alpha, and the radioiodinated analogs of PGF2 alpha may be useful as probes of the PGF2 alpha receptor.

The photochemistry of iodo, methyl and thiomethyl substituted aryl azides in toluene solution and frozen polycrystals.

The photolysis of para-methyl and para-thiomethylphenylazide at 77 K produces the corresponding triplet nitrenes which can be detected by electron paramagnetic resonance (EPR) spectroscopy. Photolysis of these azides in frozen toluene at 77 K leads to insertion of the nitrene into a benzylic C-H bond of the matrix in modest yields. Photolysis of iodinated aryl azides under these conditions does not produce triplet nitrenes that can be detected by EPR spectroscopy. In contrast to the para-methyl and para-thiomethyl substituted phenyl nitrenes, photo-induced coupling of iodo-substituted phenyl nitrenes to toluene proceeds in very poor yield.

Various 18-substituted progesterones.

In order to test the potential biological activity of 18-substituted progesterones, 3,20-dioxo-4-pregene-18-carbonitrile (ld approximately) was converted to 3,20-dioxo-4-pregnene-18-carboxylic acid (lb approximately) and methyl 3,20-dioxo-4-pregnene-18-carboxylate (ld approximately) via a sequence of reactions involving an intramolecular hydrolysis of the 18--arbonitrile. Lithium aluminum hydride reduction of the bisethylene ketal derived from la approximately furnished 18-aminomethyl-5-pregnene-3,20-dione 3,20-bisethylene ketal (8 approximately). Acetylation and hydrolysis furnished 18-acetamidomethyl-4-pregnene-3,20-dione (lf approximately) and simple hydrolysis of 8 approximately furnished 3'alpha H-5' 6'-dihydro-2',19 beta-dimethyl-3-oxo-4-goneno [13,17-c]pyridine (9 approximately). None of the compounds exhibited any activity in Clauberg or anti-Clauberg tests.

Identification of metabolites of azaperone in horse urine.

Two metabolites of the tranquilizer azaperone were extracted from alkalinized horse urine after treatment with beta-glucuronidase/sulfatase from limpets (Patella vulgata). The metabolites were identified by a combination of independent chemical synthesis and GC/MS and 1H NMR analysis. The metabolites were identified as 1-(fluorophenyl)-4-[4-(5-hydroxy-2-pyridinyl)-1-piperazinyl]-1-butanol, designated as 5'-hydroxy-azaperol, and 1-(fluorophenyl)-4-[4-(5-hydroxy-2-pyridinyl)-1-piperazinyl]-1-butanone, designated as 5'-hydroxyazaperone. A TLC screening test was developed for detecting both metabolites in basic extracts of horse urine treated with beta-glucuronidase/sulfatase. The screening test was used to detect azaperone metabolites in extracts of horse urine collected for 24 h after intravenous administration of azaperone. The administration of azaperone to horses was confirmed by GC/MS identification of 5'-hydroxyazaperone and 5'-hydroxyazaperol from basic extracts of horse urine treated with beta-glucuronidase/sulfatase. The extracted metabolites were treated with bis(trimethylsilyl)acetamide to produce trimethylsilyl (TMS) ether derivatives, and mass spectra and retention times were compared to those of the synthesized metabolites treated in the same manner.

Orientation of GTP and ADP within their respective binding sites in glutamate dehydrogenase.

Previous studies have identified the guanine and adenine binding domains of the GTP and ADP binding sites of GDH. In this study the peptide sequences within or near to the terminal phosphate-binding domains of the GTP and ADP binding sites of bovine liver glutamate dehydrogenase (GDH) were identified using photoaffinity labeling with the benzophenone nucleotide derivatives, [gamma-32P]GTPgammaBP and [gamma-32P]ATPgammaBP. Without activating light, GTPgammaBP exhibited inhibiting effects on the GDH reaction similar to GTP; ATPgammaBP, as expected, produced activating effects similar to those of ADP. Photoinsertion into GDH by both probes exhibited saturation effects in agreement with the respective kinetic effects. Specificity of labeling was supported by specific and effective reduction of photoinsertion of [gamma-32P]GTPgammaBP and [gamma-32P]ATPgammaBP into GDH by GTP and ADP, respectively. Using a combination of immobilized Fe3+-chelate affinity chromatography and reversed-phase HPLC, photolabeled peptides located within or near the phosphate-binding domains of the GTP and ADP sites were isolated. Sequence analysis showed that GTPgammaBP primarily modified a peptide near the middle of the GDH sequence, Asn135-Lys143 and Glu290-Lys295. However, ATPgammaBP modified a single peptide corresponding to the sequence Met411-Arg419 near the C-terminal domain. Using these results and the data from the previously identified base-binding domain peptides the orientation of GTP and ADP within their respective binding sites in the catalytic cleft of GDH is proposed and explained on the basis of a proposed three-dimensional schematic model structure derived from the bacterial enzyme.

Synthesis and application of bidentate photoaffinity cross-linking reagents. Nucleotide photoaffinity probes with two photoactive groups.

Two "targeted bidentate" photoaffinity cross-linking reagents, the monoanhydride of 8-N3ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]8-N3ATP gamma BP) and the monoanhydride of 8-N3GDP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]8-N3GTP gamma BP), were developed for studying the inter- and intramolecular interactions of nucleotide-binding proteins. Experiments using these bidentate reagents with two photoactive groups led to specific cross-linking: [gamma-32P]8-N3GTP gamma BP and [gamma-32P]8-N3ATP gamma BP showed intersubunit cross-linking of glutamate dehydrogenase and [gamma-32P]8-N3GTP gamma BP appeared to cross-link the alpha- and beta-subunits of tubulin. The non-azido "monodentate" versions of these reagents, the monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]ATP gamma BP) and the monoanhydride of GDP with N-(4-(benzoyl)phenylmethyl)-phosphoramide ([gamma-32P]GTP gamma BP), were also synthesized and characterized. The ability of these monodentate reagents with one photoactive group to serve as photoaffinity probes was established by photolabeling specifically the exchangeable GTP-binding domain of tubulin with [gamma-32P]GTP gamma BP and the ATP-binding domain of purified adenylate kinase and several nucleotide-binding proteins in human brain homogenate with [gamma-32P]ATP gamma BP.

Photoaffinity labeling of ribulose-1,5-bisphosphate carboxylase/oxygenase activase with ATP gamma-benzophenone. Identification of the ATP gamma-phosphate binding domain.

The phosphate-binding domain of the ATP-binding site of tobacco Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activase was elucidated by photo-affinity labeling with a monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]ATP gamma BP). Covalent incorporation of [gamma-32P]ATP gamma BP into the 42-kDa Rubisco activase subunit was dependent upon irradiation with ultraviolet light. Photolabelling of Rubisco activase with ATP gamma BP exhibited saturation kinetics; the apparent Kd for photolabeling was 5 microM. Two lines of evidence showed that ATP gamma BP modified Rubisco activase at the ATP-binding domain. First, physiological concentrations of ATP and ADP afforded complete protection against photolabeling of Rubisco activase by ATP gamma BP. Second, photolysis of Rubisco activase in the presence of ATP gamma BP decreased both the ATPase and the Rubisco activating activities. Inactivation of enzyme activity was dependent on ATP gamma BP concentration and could be prevented by including ADP during photolabeling. The region of Rubisco activase that was modified by ATP gamma BP was identified by isolating photolabeled peptides. Sequence analysis showed that ATP gamma BP modified Rubisco activase in two distinct regions; one region, S117-A136, is adjacent to the P-loop and the other region, V223-T234, exhibits homology to a region of adenylate kinase that ligates the essential metal ion. Photolabeling of these two regions of Rubisco activase was consistent with modification of the ATP gamma-phosphate-binding domain of Rubisco activase with ATP gamma BP.

The effects of calcium site occupancy and reagent length on reactivity of calmodulin lysyl residues with heterobifunctional aryl azides. Mapping interaction domains with specific calmodulin photoprobe derivatives.

The relationship of structural and functional moieties on calmodulin is important in all venues of cell activity. In this study, we investigate the effect of lysine modification on calmodulin function. Azidosalicylate reagents containing different "linker arm" lengths, between the photoactive terminus and an amine-reactive N-hydroxysuccinimidyl ester moiety were used to modify calmodulin lysines at three different positions in a calcium-dependent manner. The short cross-linker, (ASNE-2 (where ASNE represents azidosalicylate N-hydroxysuccinimidyl ester), modifies Lys-75, whereas the longer reagent, ASNE-6, modifies lysines 21, 75, and 94. The modification of these different lysines is shown to be calcium-dependent. At 1-100 microM levels of calcium, only Lys-94 is modified, suggesting that modification of this residue is directed by both the binding of calcium to calcium-binding loops III and IV and the hydrophobic pocket exposed between these two loops as a result of calcium binding. At higher calcium concentrations (> 200 microM), where sites I and II become filled, modification of Lys-21 or Lys-75 also was observed. All the modified calmodulins were able to stimulate 3',5'-cyclic-nucleotide phosphodiesterase fully although the Kact for the Lys-75 and Lys-21 derivatives increased 10- and 50-fold, respectively. None of the modifications affected the activation of erythrocyte plasma membrane Ca(2+)-ATPase. Only the ASNE-6 Lys-75 derivative showed efficient (40%) photocross-linking to the Ca(2+)-ATPase. The ASNE-2 Lys-75 derivative as well as the ASNE-6 Lys-21 and Lys-94 derivatives did not show efficient calcium-dependent photocross-linking to this enzyme.

A photoactive phosphonamide derivative of GTP for the identification of the GTP-binding domain in beta-tubulin.

A GTP photoaffinity probe (125I-APTG) was developed that incorporated an [125I]-N-(4-azidophenyl)-2-amino-3-(4-hydroxy-3-iodophenyl)propionamide group at the gamma-position of GTP through a phosphonamide linkage. A combination of saturation and GTP protection studies (90% protection at 25 microM GTP with an apparent Kd of 5 microM) validated the use of this new probe as a satisfactory GTP mimic. This probe offered the advantage of possessing an 125I radiolabel external to the GTP moiety, in contrast to the previously reported [gamma 32P]-8-N3GTP that possessed an internal 32P radiolabel. This novel feature accommodated the purification of photolabeled peptides using a combination of ion-exclusion, gel filtration, and HPLC techniques. [125I]APTG was used to identify a peptide (beta:65-79) in the exchangeable GTP-binding domain of the beta-subunit of tubulin.

Biodegradation of acyclic isoprenoids by Pseudomonas species.

The ability of various pseudomonads to utilize acyclic isoprenoids as a sole carbon source was investigated. Tests for utilization of acyclic isoprenols such as citronellol and geraniol were complicated by toxic effects of these alcohols, and most species tested were killed by exposure to citronellol or geraniol (0.1%, vol/vol) in liquid culture. In the case of Pseudomonas citronellolis, sensitivity to isoprenols is reduced by prior induction of the isoprenoid degradative pathway via either growth on succinate in the presence of citronellol or growth on citronellic acid. For this species, citronellic acid proved to be the best isoprenoid growth substrate tested. Geraniol utilization as a taxonomic indicator for different subgroups of pseudomonads is discussed. Only a few of the species tested were able to utilize acyclic isoprenoids. Two species which utilize C10 acyclic isoprenoids, P. aeruginosa and P. mendocina, were shown to contain the inducible enzyme geranyl-coenzyme A carboxylase, one of the unique enzymes in the isoprenol degradative pathway known to occur in P. citronellolis. Of the species which utilized geranitol, none showed definite growth on the homologous C15 and C20 isoprenols.

Prostaglandin photoaffinity probes: synthesis and binding affinity of C-18 substituted PGF2 alpha prostanoids bearing a perfluorinated aryl azide.

C-18 Phenoxy analogs of prostaglandin F2 alpha (PGF2 alpha) that possessed a perfluorinated aryl azide and an aryl iodide substituent were synthesized and evaluated as potential photoaffinity probes for PGF2 alpha. Prior studies indicated that only hydrophobic modifications in the omega-side chain of PGF2 alpha were compatible with high binding affinity, and this finding excluded the use of a hydroxyl-substituted C-18 phenoxy group as an activated aryl ring capable of radioiodination. Consequently, an alternate means of introducing the iodine substituent using an ipsosubstitution of a trimethylsilyl arene was developed. Although this strategy was successful from a synthetic perspective, the potential PGF2 alpha photoaffinity probe, (15S)-18-[3'-((4''-azido-2'',3'',5'',6''-tetrafluorophenyl)- methoxy) methyl-5'-iodophenoxy]-19,20-bisnorprostaglandin F2 alpha, exhibited only marginal competitive binding with [3H]-PGF2 alpha to ovine luteal cells and to plasma membranes of bovine corpora lutea. The hydrophobic but bulky C-18 substituent was presumably incompatible with effective receptor binding.

Prostaglandin photoaffinity probes: synthesis and binding affinity of aryl azide-substituted C-1 esters of prostaglandin F2 alpha.

In seeking prostaglandin F2 alpha (PGF2 alpha) photoaffinity probes possessing both an efficient, photoactive cross-linking substituent and a radiolabel of high specific activity, the synthesis and binding affinity of PGF2 alpha C-1 esters in which the alcohol component possessed either an aryl azide or a perfluorinated aryl azide was investigated. These derivatives showed great promise due to their ability to compete for the binding of [3H]-PGF2 alpha in both a luteal membrane binding assay and in a whole luteal cell binding assay. Identification of the C-1 site in PGF2 alpha as a site for modification of the PGF2 alpha molecule with photoactive alcohol derivatives represented a logical step toward the goal of developing a useful PGF2 alpha photoaffinity probe.

Synthesis and use of a biotinylated 3-azidophenothiazine to photolabel both amino- and carboxyl-terminal sites in calmodulin.

The biotinylated probe, 3-azido-10-(4-(4-biotinyl-1-piperazinyl)butyl)phenothiazine, was used to examine the phenothiazine binding domains in calmodulin (CaM) by photolabeling. This phenothiazine, synthesized from 3-azido-10-(4-(1-piperazinyl)butyl)phenothiazine and d-biotinyl tosylate, inhibited the CaM-mediated activation of phosphodiesterase (PDE) with an I50 of 12.5 (+/- 2.8) microM. Photolabeling of CaM produced covalent adducts in excellent yield (32%) in a light- and Ca2+-dependent manner. Studies performed over a range of drug concentrations suggested a 2:1 stoichiometry for the binding of the phenothiazine probe to CaM. Limited trypsin digestion and purification of the resulting fragments by either SDS-PAGE or HPLC provided two principal phenothiazine-containing peptides. Amino acid composition and sequence analyses performed on these two peptides established that both the N- and C-terminal domains in CaM, particularly the regions amino terminal to Ca2+-binding loops 1 and 3, were modified by the photoactivated phenothiazine derivative. These data, particularly for the C-terminal domain, are in excellent agreement with the X-ray structure analysis of a 1:1 CaM-trifluoperazine complex.

Synthesis and binding affinity of bidentate phenothiazines with two different photoactive groups.

The development of targeted, bidentate photoaffinity reagents for mapping the interacting domains of calmodulin (CaM) with the enzymes that it regulates required the synthesis and evaluation of the binding affinity of various phenothiazines. These photoaffinity reagents would possess a photoactive 3-azidophenothiazine group for cross-linking the hydrophobic binding domain of CaM, a second photoactive benzophenone group that would be activated at a different wavelength than the 3-azidophenothiazine group, and a suitable radiolabel. Difficulties were encountered in identifying those structural features that would be compatible with the introduction of a benzophenone group, with the solubility of these benzophenone-substituted phenothiazines, and with the ability of these phenothiazines to inhibit the calmodulin-mediated activation of phosphodiesterase. Solutions to this problem involved the preparation of phenothiazines possessing a quaternary ammonium salt, a zwitterionic amino acid, or a carbohydrate moiety. The phenothiazines that possessed photoactive 3-azido and benzophenone groups and in which one of the piperazine nitrogens in the side chain was converted to a quaternary, N-methylammonium iodide inhibited the calmodulin-mediated activation of phosphodiesterase at a level comparable to that of chlorpromazine.

Multiple classes of prostaglandin F2 alpha binding sites in subpopulations of ovine luteal cells.

A cryostorage procedure was developed to provide ovine luteal cells throughout the period of seasonal anestrus. Corpora lutea obtained from midluteal phase, superovulated ewes were dispersed enzymatically. Some dispersed cells were fractionated into subpopulations by elutriation. Dimethylsulfoxide (7.5% final concentration) in Hanks' buffered saline was added to cells at 4 degrees C, and dispersed cell preparations were frozen in a programmable cell freezer and stored at -196 degrees C. After recovery from cryopreservation, cell viability and prostaglandin F2 alpha (PGF2 alpha) binding characteristics of thawed cells were not different from those of corresponding fresh cells. Additionally, thawed cells retained the capacity to attach to culture dishes and retained responsiveness of progesterone secretion to prostaglandin E2 (PGE2) and ovine luteinizing hormone (LH), although rates of progesterone secretion were attenuated in thawed compared with fresh cells. The cryopreservation procedure will prove useful to relieve constraints in utilization of ovine luteal cells arising from reproductive seasonality in sheep. Cells retrieved from cryostorage were evaluated by studying PGF2 alpha binding characteristics. From saturation analyses (increasing amounts of radiolabeled PGF2 alpha) of PGF2 alpha binding to unfractionated cells, we detected a single class of high affinity binding sites (Kd = 17.4 +/- 2.3 nM) in addition to the nonspecific binding component. Using displacement analyses (constant radiolabeled PGF2 alpha and increasing amounts of unlabeled PGF2 alpha) and unfractionated cells, we detected additional binding sites of lower affinity (Kd = 409 +/- 166 nM) as well as the nonspecific binding component. Small luteal cells obtained by elutriation, which were essentially devoid of large cell contamination, had only low affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity heterobifunctional cross-linking reagents based on azide-substituted salicylates.

Photoaffinity heterobifunctional cross-linking reagents are described that incorporate a 4-azidosalicylate group at one terminus and an N-hydroxysuccinimidyl ester at the other terminus with a "linking arm" of variable length separating the photoactive and electrophilic termini. Exposure of calmodulin (CaM) to succinimidyl N-[2-[(4-azidosalicyloyl)oxy]ethyl]suberamate (4C) led to monoadducts at Lys-21, Lys-75, and Lys-94. Separation of the monoadducts from CaM and polyadducts, radioiodination, and photolysis in the presence of human erythrocyte plasma membrane Ca2+, Mg2(+)-ATPase led to calcium-dependent cross-linking with 8% cross-linking efficiency.

Interaction of leukotriene C4 and Chinese hamster lung fibroblasts (V79A03 cells). 1. Characterization of binding.

A novel, specific, and potent biological action of leukotriene C4 (LTC4) was demonstrated in the Chinese hamster lung fibroblast cell line V79A03 (V79 cells), namely the confirment of protection against subsequent gamma-irradiation. Consequently, studies were conducted to determine whether LTC4-conferred radioprotection could be attributed to a receptor-mediated phenomenon. Specific binding sites for leukotriene C4 (LTC4) were identified and characterized using intact V79 cells incubated at 4 degrees C in the presence of serine-borate, during which time conversion of LTC4 to LTD4 or LTE4 was undetectable. Binding was maximal in a broad region between pH 6.2 and 8.8. Ca2+, Mg2+, and Na+ were not required for binding, and binding was not altered by GTP, ATP, or cAMP, by leukotrienes B4, D4, or E4, or by the leukotriene end point antagonists LY 171883, FPL 55712, or Revlon 5901-5. Scatchard analyses and kinetic experiments indicated the presence of high-affinity [Kd = 2.5 +/- 0.63 nM, approximately 9.9 x 10(5) sites/cell] and low-affinity [Kd = 350 +/- 211 nM, approximately 2.7 x 10(6) sites/cell] binding sites. The observed binding characteristics of LTC4 to V79 cells are consistent with a receptor-mediated phenomenon. In a companion communication which follows this report, we report the subcellular distribution of LTC4 binding to V79 cells and demonstrate that this binding is unlikely to be attributed principally to interaction with glutathione-S-transferase.

Interaction of leukotriene C4 and Chinese hamster lung fibroblasts (V79A03 cells). 2. Subcellular distribution of binding and unlikely role of glutathione-S-transferase.

It was reported previously that radiation-induced cytotoxicity in V79A03 (V79) cells was attenuated by pretreatment of cells with leukotriene C4 (LTC4), leading us to determine that V79 cells possessed specific binding sites, with characteristics of receptors, for LTC4 (see the preceding, companion communication). Additional studies were conducted to determine the subcellular distribution and the chemical nature of the LTC4 binding site in V79 cells. Trypsin treatment of cells before LTC4 binding assays resulted in a 74% reduction in high-affinity binding. In tests to examine the subcellular location of LTC4 binding, plasma membrane and nuclear fractions were obtained from V79 cells. In contrast to Scatchard analyses of LTC4 binding to intact cells which were curvilinear, Scatchard analyses of nuclear and plasma membrane fractions were linear, indicative of the presence in these cellular substituents of low and high-affinity binding, respectively. To examine the nature of the high-affinity LTC4 binding sites, intact V79 cells were photolyzed with [3H]-LTC4 rendered photoactive by preincubation with N-hydroxysuccinimidyl-4-azidobenzoate. The cell-bound radioactivity migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of approximately 40 kdal. Five different commercial preparations of glutathione-S-transferase (GST), which has been implicated as a source of LTC4 "specific binding" in other cells, migrated in the same SDS-PAGE system with an apparent molecular weight of 20-24 kdal. Furthermore, preincubations of V79 cells with three antisera generated against GST had minimal effects upon subsequent LTC4 binding to intact cells. These data, taken together with the data from the preceding companion communication, suggest that the radioprotective effect of LTC4 upon V79 cells may be attributable to a receptor-mediated phenomenon which appears distinct from leukotriene binding to GST.