Predicting XAFS scattering path cumulants and XAFS spectra for metals (Cu, Ni, Fe, Ti, Au) using molecular dynamics simulations.

The ability of molecular dynamics (MD) simulations to support the analysis of X-ray absorption fine-structure (XAFS) data for metals is evaluated. The low-order cumulants (ΔR, σ(2), C3) for XAFS scattering paths are calculated for the metals Cu, Ni, Fe, Ti and Au at 300 K using 28 interatomic potentials of the embedded-atom method type. The MD cumulant predictions were evaluated within a cumulant expansion XAFS fitting model, using global (path-independent) scaling factors. Direct simulations of the corresponding XAFS spectra, χ(R), are also performed using MD configurational data in combination with the FEFF ab initio code. The cumulant scaling parameters compensate for differences between the real and effective scattering path distributions, and for any errors that might exist in the MD predictions and in the experimental data. The fitted value of ΔR is susceptible to experimental errors and inadvertent lattice thermal expansion in the simulation crystallites. The unadjusted predictions of σ(2) vary in accuracy, but do not show a consistent bias for any metal except Au, for which all potentials overestimate σ(2). The unadjusted C3 predictions produced by different potentials display only order-of-magnitude consistency. The accuracy of direct simulations of χ(R) for a given metal varies among the different potentials. For each of the metals Cu, Ni, Fe and Ti, one or more of the tested potentials was found to provide a reasonable simulation of χ(R). However, none of the potentials tested for Au was sufficiently accurate for this purpose.

Lift-off protocols for thin films for use in EXAFS experiments.

Lift-off protocols for thin films for improved extended X-ray absorption fine structure (EXAFS) measurements are presented. Using wet chemical etching of the substrate or the interlayer between the thin film and the substrate, stand-alone high-quality micrometer-thin films are obtained. Protocols for the single-crystalline semiconductors GeSi, InGaAs, InGaP, InP and GaAs, the amorphous semiconductors GaAs, GeSi and InP and the dielectric materials SiO2 and Si3N4 are presented. The removal of the substrate and the ability to stack the thin films yield benefits for EXAFS experiments in transmission as well as in fluorescence mode. Several cases are presented where this improved sample preparation procedure results in higher-quality EXAFS data compared with conventional sample preparation methods. This lift-off procedure can also be advantageous for other experimental techniques (e.g. small-angle X-ray scattering) that benefit from removing undesired contributions from the substrate.

Identification of Vinca alkaloid acceptors in P388 murine leukemia cells with a photoactive analogue of vinblastine.

The cytotoxic, antimitotic, and growth inhibition properties of a photoactive analogue of vinblastine, N-(p-azidobenzoyl)-N'-beta-aminoethylvindesine (NABV), and vinblastine on P388 murine leukemia cells were compared. After 72-h exposure, the 50% drug-inhibitory concentrations for exponentially growing P388 leukemic cells were 1.2 nM for NABV and 0.6 nM for vinblastine. The ultrastructural effects of NABV and vinblastine on P388 cells were similar: formation of tubulin paracrystals; mitotic arrest (C-mitosis); increased post-C-mitotic multinucleated cells; increased number of annulate lamellae; and the appearance of intracytoplasmic paired cisternae. [3H]NABV was used to identify Vinca alkaloid binding sites in P388 cells by photoaffinity labeling. After irradiation at 302 nm, radioactive Vinca alkaloid binding components were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified in 1-mm gel slices. The most prominent photolabeled species were Mr 44,000, 54,000, and 75,000 polypeptides located in the 100,000 X g supernatant fraction. The Mr 54,000 component was also observed in the membrane fraction. Specific photolabeling of Mr 54,000 and 44,000 polypeptides was blocked in the presence of 20 microM excess of vinblastine and was saturable with half-maximal saturation concentrations of 0.18 and 0.4 microM [3H]NABV, respectively. The Mr 54,000 component was identified as a tubulin subunit by immunoprecipitation with antitubulin monoclonal antibodies. Since NABV and vinblastine have similar pharmacological and biological properties, this photoactive analogue may be useful for identifying important Vinca alkaloid cellular acceptors which may be responsible for drug cytotoxic and antineoplastic activities.

Anthracycline photoaffinity labeling of a mitochondrial polypeptide in P388 murine leukemic cell lines.

N-(p-Azido[3,5-3H]benzoyl)daunorubicin ([3H]NABD), a radioactive photoactive anthracycline analogue, was used to photoaffinity label anthracycline binding polypeptides in P388 murine leukemic cell lines. Whole cell homogenates were mixed with 6 X 10(-8) M [3H]NABD, exposed to ultraviolet light, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for radiolabel incorporation. Autoradiofluorography showed incorporation of radioactivity into a Mr 18,000 component independent of polypeptides prominently stained with Coomassie blue. Photolabeling of subcellular fractions showed predominant mitochondrial localization of the Mr 18,000 radiolabel. The protein composition of the photolabeled constituents was confirmed by treatment with proteinase K, DNase and RNase, or by lipid extraction with organic solvent. [3H]NABD photolabeling of homogenates from anthracycline sensitive and resistant cells resulted in Mr 18,000 radiolabel incorporation of 3,966 +/- 355 and 6,487 +/- 533 dpm per 50 micrograms cellular protein for anthracycline sensitive and resistant cells, respectively (P less than 0.005). These studies characterize the photoaffinity labeling of a low molecular weight mitochondrial polypeptide using a photoactive anthracycline analogue. The role for this polypeptide as a mediator of anthracycline activity remains to be determined.

Cell surface membrane protein changes during the differentiation of cultured human promyelocytic leukemia HL-60 cells.

The human promyelocytic leukemia cell line HL-60 was induced to differentiate in vitro by treatment with dimethyl sulfoxide or retinoic acid. Morphological maturation was accompanied by a total loss of transferrin binding and a 7-fold increase in the percentage of cells reducing nitro blue tetrazolium. Cell surface membrane proteins and glycoproteins were labeled with 125I by the lactoperoxidase-H2O2 or 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (Iodo-Gen) methods and analyzed by two-dimensional isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A minimum of 12 cell surface proteins were unchanged, 3 proteins (Mr 95,000, 87,000, and 77,000) were lost, and up to 7 new proteins (Mr 270,000, 240,000, 150,000, 135,000, 58,000, 56,000, and 50,000) appeared during HL-60 cell differentiation. The kinetics of disappearance of one major labeled cell surface protein (Mr 95,000) within two days during treatment with retinoic acid correlated with the loss of cellular transferrin binding. This protein was identified as the transferrin receptor by affinity absorption of extracts of 125I-surface protein-labeled cells to transferrin-Sepharose beads. The affinity-purified component had molecular weights of 190,000 and 95,000 under nonreducing and reducing conditions, respectively, confirming its dimeric structure. Two-dimensional electrophoresis of cell surface membrane-labeled proteins of normal human granulocytes confirmed the absence of the transferrin receptor and identified cell surface proteins with molecular weight and pI values corresponding to three of the new cell surface proteins which appeared during HL-60 maturation. The most intensely labeled of these had a molecular weight of about 55,000, and was confirmed as being identical to the corresponding Mr 58,000 HL-60 cell surface membrane protein by one-dimensional peptide-mapping analysis. This prominent new Mr 55,000 to 58,000 protein increased continuously throughout retinoic acid-induced maturation and was identified as a major terminal myeloid differentiation cell surface membrane protein.

Induction of a human carbonyl reductase gene located on chromosome 21.

Carbonyl reductase (EC 1.1.1.184) belongs to the group of enzymes called aldo-keto reductases. It is a NADPH-dependent cytosolic protein with specificity for many carbonyl compounds including the antitumor anthracycline antibiotics, daunorubicin and doxorubicin. Human carbonyl reductase was cloned from a breast cancer cell line (MCF-7). The cDNA clone contained 1219 base paires with an open reading frame corresponding to 277 amino acids encoding a protein of Mr 30,375. Southern analysis of genomic DNA digested with several restriction enzymes and analyzed by hybridization with a labeled cDNA probe indicated that carbonyl reductase is probably coded by a single gene and does not belong to a family of structurally similar enzymes. Southern analysis of 17 mouse/human somatic cell hybrids showed that carbonyl reductase is located on chromosome 21. Carbonyl reductase mRNA could be induced 3-4-fold in 24 h with 10 microM 2,(3)-t-butyl-4-hydroxyanisole (BHA), beta-naphthoflavone or Sudan 1.

Synthesis and characterization of inhibitors of myristoyl-CoA:protein N-myristoyltransferase.

Several substrate and product analogs were synthesized and tested as in vitro inhibitors of bovine brain N-myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97). At 40 microM, the acyl CoA analog, S-(2-ketopentadecyl)-CoA, completely inhibited NMT in the presence of 80 microM myristoyl CoA. Decreasing but marked inhibition was also observed with the acyl CoA analogs, S-(2-bromo-tetradecanoyl)-CoA and S-(3-(epoxymethylene)dodecanoyl)-CoA, and the multisubstrate derivative N-(2-S-CoA-tetradecanoyl)glycinamide in the presence of 40 microM myristoyl CoA. Inhibition was also observed with the non-coenzyme A myristoyl analog, 1-bromo-2-pentadecanone. All of the above compounds exhibited reversible competitive inhibition kinetics with respect to myristoyl CoA with Ki values of 0.11 to 24 microM. Two additional acyl CoA analogs, S-(cis-3-tetradecenoyl)-CoA and S-(3-tetradecynoyl)-CoA, functioned as alternative substrates for NMT.

Identification and characterization of multiple forms of bovine brain N-myristoyltransferase.

N-Myristoyltransferase (NMT) catalyzes the co-translational addition of myristic acid to the N-terminal glycine of many cellular, viral, and fungal proteins which are essential to normal cell functioning and/or are potential therapeutic targets. We have found that bovine brain NMT exists as a heterogeneous mixture of interconvertible high molecular mass multimers involving approximately 60-kDa NMT subunit(s). Gel filtration chromatography of partially purified NMT at low to moderate ionic strength yields NMT activity eluting as 391 +/- 52 and 126 +/- 17 kDa peaks as well as activity which profiles the protein fractions and likely results from NMT nonspecifically associating with background proteins and/or column matrix. Chromatography in 1 M NaCl causes 100% of this activity to elute as a single peak of approximately 391 kDa. Subsequent treatment of the approximately 391 kDa activity peak with an NMT peptide reaction product (i.e. N-myristoyl-peptide) results in approximately 75% of the activity re-eluting as a approximately 126-kDa peak in 1 M NaCl. Rechromatography also yields small amounts of a approximately 50-kDa NMT monomer which increases with prior storage at 4 degrees C. Up to 5 NMT subunits were identified by SDS-polyacrylamide gel electrophoresis and specific immunoblotting with a human NMT peptide antibody and by cofactor-dependent chemical cross-linking with an 125I-peptide substrate of NMT. The prominent 60 kDa and minor 57-, 53-, 49-, and 47-kDa NMT immunoblotted subunits co-migrate with five of nine silver-stained proteins in an enzyme preparation purified > 7,000-fold with approximately 50% yield by selective elution from octyl-agarose with the myristoyl-CoA analog, S-(2-ketopentadecyl)-CoA. Storage at 4 degrees C also leads to conversion of the larger NMT subunit(s) into 49 and 47 kDa forms with no loss of NMT activity. These results identify two interconvertible forms of NMT in bovine brain that result from NMT subunit multimerization and/or complex formation with other cellular proteins. The data also identify a fully active NMT monomer which arises from subunit proteolysis. This study thus reveals a previously unappreciated level of NMT complexity which may have important mechanistic and/or regulatory significance for N-myristoylation in mammalian cells.

N-myristoylation of p60src. Identification of a myristoyl-CoA:glycylpeptide N-myristoyltransferase in rat tissues.

A 16-residue synthetic peptide corresponding to the N-terminal sequence of p60src was used as the acyl acceptor in an assay for myristoyl-CoA:glycylpeptide N-myristoyltransferase in rat tissues. An additional C-terminal tyrosine amide was added to this peptide to facilitate radioiodination and enhance detectability. Reverse-phase h.p.l.c. enabled the simultaneous detection and quantification of the peptide substrate and its N-myristoylated product. N-Myristoyltransferase activity was highest in the brain with decreasing activities in lung, small intestine, kidney, heart, skeletal muscle and liver. Brain activity was distributed approximately equally between the 100,000 g pellet and supernatant fractions. The soluble enzyme exhibited a Kappm of 20 microM for the src peptide and an optimum between pH 7.0 and 7.5. Maximum N-acylating activity was seen with myristoyl (C14:0)-CoA with lower activities found with the C10:0-CoA and C12:0-CoA homologues. No activity was obtained with palmitoyl (C18:0)-CoA but this derivative inhibited N-myristoyltransferase activity greater than 50% at equimolar concentrations with myristoyl-CoA. With a decapeptide corresponding to the N-terminal sequence of the cyclic AMP-dependent protein kinase catalytic subunit as the acyl acceptor, the brain enzyme displayed a Kapp.m of 117 microM and was about 14-fold less catalytically effective than with the p60src acyl acceptor. Transferase activity was also seen with a 16-residue peptide corresponding to the N-terminal sequence of the HIV p17gag structural protein. Inhibition studies with shorter src peptide analogues indicated an enzyme specificity for the p60src acyl acceptor beyond 9 residues.

Human N-myristoyltransferase amino-terminal domain involved in targeting the enzyme to the ribosomal subcellular fraction.

N-Myristoyltransferase (NMT) catalyzes the cotranslational acylation with myristic acid of the NH2-terminal glycines of a number of cellular and viral proteins. Most of the in vitro NMT activity (60-85%) in isoosmotic cell homogenates of human lymphoblastic leukemia (i.e. CEM and MOLT-4) and cervical carcinoma (i.e. HeLa) cells was shown to be associated with the ribosomal subcellular fractions by differential centrifugation. Also found in the ribosomal fractions was a approximately 60-kDa protein that was specifically immunoblotted with an anti-human NMT (hNMT) peptide antibody. This approximately 60-kDa protein was stable in the presence of proteolytic enzyme inhibitors but was gradually converted into a approximately 46-kDa species when stored in the absence of protease inhibitors. Sucrose density gradient centrifugation of the ribosomal fraction resulted in the hNMT activity sedimenting exactly coincident with the 260 nm absorption profile and exhibiting A260/A280 absorption ratios >1.8, indicating an association of NMT with putative ribosomal particle(s)/subunit(s). The subcellular targeting of hNMT was also examined by immunoblotting subcellular fractions from HeLa cells transfected with plasmids containing FLAG epitope-tagged hNMT inserts corresponding either to the originally assigned hNMT gene or to an alternative open reading frame initiated from an in-frame start site upstream from the assumed hNMT start site. Anti-FLAG immunoblotting of cells transfected with a plasmid containing the larger insert revealed FLAG-NMT primarily in the ribosomal fraction with an apparent molecular mass similar to the approximately 60-kDa native hNMT. In contrast, immunoblotting of cells transfected with a plasmid containing the smaller insert identified a approximately 50-kDa FLAG-NMT predominantly in the cytosolic fraction. An analysis of mixtures of CEM ribosomes and serial dilutions of purified recombinant FLAG-NMTs demonstrated that the approximately 60-kDa FLAG-NMT binds ribosomes with higher affinity than the approximately 50-kDa FLAG-NMT. These in vivo and in vitro subcellular targeting and recombinant expression experiments identify a native hNMT that is 10-12 kDa larger than the enzyme predicted by the originally assigned hNMT gene and which is apparently translated from an alternative up-stream start site. The data also indicate that although the unique NH2-terminal residues encoded by this larger open reading frame are not required for in vitro catalytic activity, they do provide signal(s) involved in targeting hNMT to the ribosomal subcellular fraction where cotranslational N-myristoylation occurs.

Rat heart anthracycline-binding polypeptides identified by photoaffinity labeling.

A radioactive, photoactive anthracycline analogue, N-(p-azido-[3,5-3H]benzoyl)-daunorubicin (3H-NAB-daunorubicin), was synthesized and characterized by UV-visible absorption and infrared analyses. 3H-NAB-daunorubicin photoaffinity labeling of rat heart homogenates resulted in the identification of two prominently radiolabeled anthracycline-binding polypeptides of 18.3 and 31.2 kDa. Photoaffinity labeling with photoactive doxorubicin (Adriamycin), carminomycin, and nonanthracycline model compounds resulted in a clear structural dependence for binding to the 18.3-and 31.2-kDa species. In the presence of daunorubicin or N-substituted daunorubicin analogues, 3H-NAB-daunorubicin photolabeling of the 18.3-kDa polypeptide was inhibited. Photolabeling was dependent on time of UV light exposure and protein concentration and was unaffected by the presence of nitrene scavengers. The 18.3-kDa polypeptide photolabeling was saturable and reversed by greater than 90% in the presence of a 16-fold molar excess of nonradioactive analogue. Photolabeling of heart subcellular fractions demonstrates that both the 18.3- and 31.2-kDa polypeptides were localized to the inner mitochondrial membrane. Since the anthracyclines are known to have several effects on heart mitochondrial function, the identification of specific polypeptide acceptors using photoactive anthracycline analogues may elucidate biochemical mechanisms of anthracycline cellular activity.

Bacterial expression, purification, and in vitro N-myristoylation of HIV-1 p17gag.

The coding region of the N-terminal 17-kDa portion of HIV-1 Pr55gag (p17gag) was cloned into the pET-3c expression vector and was used to overexpress HIV-1 p17gag in Escherichia coli. Induction of the transformed bacteria caused the accumulation of a 17-kDa polypeptide in the soluble cell fraction which was released by sonication in hypotonic nondetergent buffer. The 17-kDa polypeptide was purified by ammonium sulfate precipitation and successive chromatography on G-75 Sephadex, DEAE-Sephacel, and S-Sephadex. The final product was purified 12-fold with about a 16% recovery from the original soluble cell lysate and was judged to be 97+% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blotting with two different antibodies confirmed the identify of the purified 17-kDa polypeptide as authentic p17gag. In the presence of myristoyl-CoA and bovine brain N-myristoyl-transferase, p17gag was quantitatively N-myristoylated in vitro with a pseudo-first-order rate constant of 4.7 +/- 1.0 x 10(-3) min-1, but with only about 3% of the catalytic efficiency of N-myristoylation of a 16-residue peptide homologous to the N-terminus of p17gag. The myristate group in the N-myristoylated p17gag was stable to treatment with detergent and hydroxylamine consistent with a covalent N-acyl-amide linkage. The N-myristoylglycyl linkage was confirmed by partial acid hydrolysis and identification of the p-nitrobenzylazlactone derivative of the resulting N-myristoylglycine by high-performance liquid chromatography.

Reductive activation of mitomycin C and mitomycin C metabolites catalyzed by NADPH-cytochrome P-450 reductase and xanthine oxidase.

Under anaerobic conditions and with proper electron donors, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and xanthine oxidase (EC 1.2.3.2) similarly reductively metabolized mitomycin C. Reversed phase high performance liquid chromatography was used to separate, detect, and isolate several metabolites. Three metabolites were identified by mass spectrometry and thin layer chromatography as 1,2-cis- and trans-2,7-diamino-1-hydroxymitosene and 2,7-diaminomitosene. Three metabolites were phosphate-dependent, and two of them were identified to be 1,2-cis- and trans-2,7-diaminomitosene 1-phosphate. The amounts of the five identified metabolites generated during the reduction of mitomycin C varied with pH and nucleophile concentration. At pH 6.5, 2,7-diaminomitosene was essentially the only metabolite formed, whereas from pH 6.8 to 8.0, trans- and cis-2,7-diamino-1-hydroxymitosene increased in quantity as 2,7-diaminomitosene decreased. The disappearance of mitomycin C and the production of metabolites were enzyme and mitomycin C concentration-dependent. Substrate saturation was not reached for either enzyme up to 5 mM mitomycin C. Electron paramagnetic resonance studies demonstrated the formation of mitomycin C radical anion as an intermediate during enzymatic activation. Our results indicate that either enzyme catalyzed the initial activation of mitomycin C to a radical anion intermediate. Subsequent spontaneous reactions, including the elimination of methanol and the opening of the aziridine ring, generate one active center at C-1 which facilitates nucleophilic attack. Simultaneous generation of two reactive centers was not observed. All five primary metabolites were metabolized further by either flavoenzyme. The secondary metabolites exhibited similar changes in their absorbance spectra and were unlike the primary metabolites, suggesting that a second alkylating center other than C-1 was generated during secondary activation. We propose that secondary activation of monofunctionally bound mitomycin C is probably a main route for the bifunctional binding of mitomycin C to macromolecules and that the cytotoxic actions of mitomycin C result from multiple metabolic activations and reactions.

Surface membrane glycoproteins of wild-type and differentiation-inducer-resistant HL-60 cells.

Surface membrane glycoproteins (SMGs) of cells from the parental wild-type HL-60 cell line and from three sublines variably cross-resistant to the granulocyte differentiation-inducing effects of retinoic acid (RA), dimethylsulfoxide (DMSO), and certain purine bases (6-thioguanine [6TG] or hypoxanthine) were studied by one-dimensional and two-dimensional gel electrophoresis. After both oligosaccharide (periodate/borotritide) and peptide (1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycouril) ectolabeling procedures, striking common changes were noted in the gel electrophoretic patterns of the SMGs from the RA- and 6TG-resistant sublines compared to those from the wild-type HL-60 line or the DMSO-resistant subline. Most prominently, this included the presence in the RA- and 6TG-resistant cells of an apparent high molecular weight acidic glycoprotein(s) (mol wt, 200 to 285 kilodaltons [kD]; isoelectric point range [pl], 4.5 to 6.0) not observed in the wild-type or DMSO-resistant cells and, conversely, the presence of a lower molecular weight glycoprotein(s) (mol wt, 120 to 165 kD; pl, 4.2 to 5.9) in the wild-type and DMSO-resistant cells, which was absent or much reduced in the RA- and 6TG-resistant cells. These acidic SMGs did not change as a function of the induction of granulocyte differentiation. However, some other more basic SMGs varied as a function of granulocyte differentiation in both the wild-type and differentiation inducer-resistant sublines, including the loss of the transferrin receptor and the gain of a mol wt 55- to 60-kD neutrophil-associated protein. In the context of previously reported information, these results indicate (1) that the overall pattern of SMG changes in the RA- and 6TG-resistant cells closely resembles that associated with multidrug (pleiotropic) resistance to cytotoxic agents in a variety of mammalian cells; (2) that the RA/6TG resistance-associated SMG changes are not granulocyte differentiation stage-specific; and (3) that either the RA/6TG resistance-associated SMG changes are not related to the resistance mechanism or they are involved in the resistance/cross-resistance mechanism(s) for RA/purine bases but not for DMSO.

Rheological properties of fibrin clots. Effects of fibrinogen concentration, Factor XIII deficiency, and Factor XIII inhibition.

The effect of fibrinogen concentration, Factor XIII deficiency, and Factor XIII inhibition, utilizing hydroxylamine, on the formation of clot structure in vitro was studied in human platelet-free plasma systems. Rheological and biochemical techniques were employed to relate changes in clot elasticity and viscosity to clot structure formation following recalcification of citrate anticoagulated samples. Classical theories of linear viscoelasticity for swollen crosslinked materials were shown to give an excellent estimate of the number of covalent crosslinks per fibrin monomer, which is directly attainable from rheological data. SDS gel electrophoresis was utilized to show qualitatively that decreases in maximum clot elasticity, at constant fibrinogen concentration, are directly related to a decrease in Factor XIII-mediated intermolecular crosslinking. The use of the technique to investigate both kinetic and equilibrium crosslinking (or structure formation) abnormalities in plasma systems is discussed.

Effects of indole alkaloids on multidrug resistance and labeling of P-glycoprotein by a photoaffinity analog of vinblastine.

Multidrug resistant cells are characterized by decreased drug accumulation and retention, thought to be mediated by a high molecular weight glycoprotein, P-glycoprotein (P-gp). Agents such as verapamil have been shown to increase anticancer drug cytotoxicity and increase the amount of drug accumulated and retained by such cells. We show here that in addition to verapamil, reserpine, chloroquine, quinine, quinacrine, yohimbine, vindoline, and catharanthine also enhance the cytotoxicity of vinblastine (VLB) in a multidrug resistant, human leukemic cell line, CEM/VLB1K, described here for the first time. These cells express P-gp as a doublet that is photoaffinity labeled by the analog of VLB, N(p-azido-[3-125I]salicyl)-N'-beta-aminoethylvindesine ([125I]NASV). Both reserpine and, to a lesser extent, verapamil, compete with [125I]NASV for binding to P-gp. We also found that chloroquine, quinacrine, vindoline, and catharanthine, each of which enhanced VLB cytotoxicity in CEM/VLB1K cells by 10- to 15-fold, similarly inhibited [125I]NASV labeling of P-gp. However, neither quinine nor yohimbine inhibited this labeling, and the inhibition produced by catharanthine and vindoline was the greatest or exclusively on the lower band of the P-gp doublet. Our results suggest a complex relationship between the ability of a compound to modulate MDR and its ability to compete for binding to P-gp.

Vinblastine photoaffinity labeling of a high molecular weight surface membrane glycoprotein specific for multidrug-resistant cells.

Photoactive radioactive analogues of vinblastine were used to photoaffinity label membranes of Chinese hamster lung drug-sensitive (DC-3F), multidrug-resistant sublines selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/ADX), and revertant (DC-3F/ADX-U) cells. A radiolabeled doublet (150-180 kDa) consisting of a major and minor band which was barely detectable in parental drug-sensitive cells was increased up to 150-fold in the drug-resistant variants but only 15-fold in the revertant cells. Photoaffinity labeling in the presence of 200-fold excess vinblastine reduced radiolabeling of the 150-180-kDa species up to 96%, confirming its Vinca alkaloid binding specificity. The radiolabeled doublet comigrated with a Coomassie Blue stained polypeptide doublet in the drug-resistant cells and was immunoprecipitated with polyclonal antibody which is specific for the 150-180-kDa surface membrane glycoprotein in multidrug-resistant cell lines. The identification of this Vinca alkaloid acceptor in multidrug-resistant plasma cell membranes suggests the possibility of a direct functional role for the 150-180-kDa surface membrane protein in the development of multidrug resistance.