The role of the BCG dose and the mouse strain in the inhibition of development of neoplasms susceptible and nonsusceptible to the action of natural cytotoxic cells

The susceptibility of Ehrlich Ascites Carcinoma (EAC) cells to the action of natural cytotoxic cells of DBA/2 and Balb/c mice in vitro was established. Leukaemia L 1210 cells proved insensitive to the in vitro action of natural cytotoxic cells of DBA/2 mice, but not to those of Balb/c ones. BCG, one of the inductors of cytotoxic NK cells, when administered to DBA/2 or Balb/c mice before introduction of EAC cells inhibited the growth of this tumour but did not retard the development of leukaemia L 1210 in DBA/2 mice. The change in the number of peritoneal exsudate cells (PEC) in DBA/2 mice after intraperitoneal injection of BCG was demonstrated to be dependent on the dose and the time elapsed after bacilli introduction. The antitumour action of BCG does not depend on changes in the number of PEC caused by the bacilli. Both large (3.0 mg) and small (0.02 mg) doses of BCG inhibit the development of EAC in Balb/c mice ("sensitive" to BCG), notwithstanding the time of administration of the bacilli. In DBA/2 mice ("resistant" to BCG) development of EAC can be inhibited only by the large dose of BCG since small one is sometimes ineffective.     

Development of host-dependent high-grade tumor-specific immunity through a novel mechanism triggered by the Lyt-2+ tumor-specific T cell clone (K7L) that induces temporal growth of L1210 leukemia-K7L-variant

When a murine leukemia L1210-specific Lyt-2+ T cell clone, K7L, was injected i.p. into CD2F1 mice together with L1210, the normal growth of L1210 in the peritoneal cavity of the mice at the early stage (days 0 to 5) was strongly inhibited, but L1210 grew progressively at the middle-stage (days 5 to 10), and then was rejected at the late stage (days 10 to 20). The mice thus survived for long times (more than 60 days), whereas the normal control injected with L1210 alone died within 14 days. The L1210 that grew at the middle stage in mice initially inoculated with L1210 together with K7L was a K7L-insensitive (K7L-) variant. All of eight tumor clones established from L1210-K7L- by limiting dilution was insensitive to the antitumor activity of K7L, and this property of tumor clones was stable after repeated in vitro passage. The initial depression of the L1210 growth by K7L followed by growth and rejection of the variant L1210-K7L- by the host T cell activity was then found to prepare a strong, long-lasting (more than 3 mo) immunity to protect mice against the high-dose (10(7) cells per mouse) challenge of original L1210. Corresponding to this result, definite tumor (L1210)-specific cytotoxic T lymphocyte (CTL) activity against both variant and original L1210 targets was developed by antigen (L1210) restimulation in the culture of spleen cells from these mice, but was not increased to a detectable level before L1210-K7L- variant started to grow. It was suggested that the 1210-K7L- variant and the original L1210 should have the common tumor-specific antigen that was independent of the K7L-reactive antigen, and that original L1210, whose growth was retarded by K7L, primed the host with the common antigen to be enormously boosted by the subsequently growing L1210-K7L- variant.     

Enhanced antitumor activity [correction for activitiy] of trans(+/-)-1,2-diaminocyclo- hexaneglutamatoplatinum(II) formulated with stealth liposome

The antitumor platinum(II) compound, [Pt(dach)(Glu)] (dach=trans(+/-)-1,2-diaminocyclohexane, Glu=glutamate) was formulated with a stealth liposome to improve its biological activity. Liposomes were composed of PC/PEG2000-PE/CH (PC=1,2-diacyl-glycero-3-phosphocholine; PEG2000-PE=poly(ethylene glycol)2000-1,2-diacyl-glycero-3-phosphoethanolamine; CH=cholesterol) involving different acyl moieties of phospholipids such as DO (dioleoyl), DM (dimyristoyl) or DS (distearoyl) group. Among the different acyl groups in the stealth liposomes, the DM formulation was optimal for the preparation of the liposomal [Pt(dach)(Glu)] at the mole ratio of DMPC/PEG2000-DMPE/CH=50/5/45 and at the weight ratio of drug/lipid=1/20, which is represented as L-[Pt(dach)(Glu)]. In vitro cytotoxicity was examined in sensitive A2780 and ME180 and their cisplatin-resistant A2780/PDD and ME180/PDD cancer cells. L-[Pt(dach)(Glu)] was 2 approximately 3 times more cytotoxic than the free complex [Pt(dach)(Glu)] and cisplatin in sensitive cells, and 4 approximately 8 times more cytotoxic in resistant cells. Thus, the resistance index of L-[Pt(dach)(Glu)] was 1.3 approximately 2 while those of the free complex and cisplatin were 5 approximately 6, which indicates that L-[Pt(dach)(Glu)] overcome the cisplatin resistance in both resistant cells. In vivo antitumor activity was assayed against the L1210/S leukemia. The optimal activities (% T/C) of the free complex and L-[Pt(dach)(Glu)] were >459/20 and >442/200 mg/kg, respectively. Considering the amount of the platinum complex in L-[Pt(dach)(Glu)], the liposomal [Pt(dach)(Glu)] displayed 2-fold higher drug potency than the free complex. The biodistribution experiment using LE52 tumor-bearing mouse showed excellent lung targeting property of L-[Pt(dach)(Glu)].     

Prostaglandin E1 receptor from mouse mastocytoma P-815 cells couples to 60 kDa GTP-binding protein.

The stable [3H]prostaglandin E1 (PGE1)-bound receptor, which couples to 60 kDa GTP-binding protein, from membranes of mouse mastocytoma P-815 cells has been purified and characterized. When the membranes were preincubated with [3H]PGE1 for 60 min at 37 degrees C, the dissociation of the ligand from the receptor was remarkably decreased, even in the presence of GTP gamma S. The stable [3H]PGE1-bound receptor complex was solubilized with 6% digitonin. The solubilized [3H]PGE1 receptor was eluted with [35S]GTP gamma S bindings activity from an Ultrogel AcA44 column. The fractions containing activities of both [3H]PGE1 and [35S]GTP gamma S bindings were further purified by column chromatographies on wheat germ agglutinin (WGA)-agarose and phenyl-Sepharose CL-4B. The partially purified [3H]PGE1-bound receptor was affinity-labeled with [14C]5'-p-fluorosulfonylbenzoylguanosine and a protein with a molecular mass of 60 kDa was detected. These results suggest that the ligand-bound PGE1 receptor of P-815 cells associates with a novel GTP-binding protein with a molecular mass of 60 kDa.     

Mice with Spontaneous Mammary Tumors Develop Type-Specific Neutralizing and Cytotoxic Antibodies Against the Mouse Mammary Tumor Virus Envelope Protein gp52

Sera from C3H mammary tumor-bearing mice contain cytotoxic antibodies for mouse mammary tumor virus (MMTV)-producing cells, based on (51)Cr release in a complement-dependent serum cytotoxicity assay. The cytotoxic antibodies could be absorbed by purified C3H MMTV gp52 and C3H MMTV-infected cat cells (C3H [MMTV] CrFK) containing cell surface MMTV gp52. However, purified MMTV p27 and uninfected CrFK cat cells were negative. Absorption of the sera with GR (MMTV) CrFK cells also removed all of the cytotoxicity, whereas absorption with RIII (MMTV) CrFK cells was negative, even though all three infected cat cells contained equivalent amounts of gp52. The same C3H cytotoxic sera also neutralized the focus-forming capacity of a C3H MMTV pseudotype of Kirsten sarcoma virus containing MMTV gp52. In contrast, sera from mammary tumor-bearing GR and RIII mice did not neutralize the pseudotype. Furthermore, neutralization could be achieved only by anti-gp52 but not by anti-gp36, -p27, -p14, or -p10 C3H MMTV sera. The gp52's of C3H, GR, and RIII MMTV could also be distinguished by using a type-specific competition radioimmunoassay employing (125)I-gp52 of C3H MMTV and a hyperimmune rabbit anti-C3H MMTV serum. To demonstrate these differences directly, we studied the primary structure of gp52 on the surface of the C3H, GR, and RIII (MMTV) CrFK cells. Two-dimensional tryptic peptide maps of the cell surface lactoper-oxidase-catalyzed iodinated gp52's revealed a greater number of peptides common to the gp52's of C3H and GR MMTVs than to RIII MMTV gp52. These results demonstrate that gp52 is a major target antigen for both cytotoxic and neutralizing antibodies, that the cell surface and virion-associated gp52's of C3H, GR, and RIII MMTV contain both group- and type-specific determinants, and that C3H and GR MMTV gp52's are antigenically more related to each other than to RIII MMTV gp52. Furthermore, C3H mammary tumor-bearing mice develop type-specific antibodies capable of recognizing unique gp52 determinants and, therefore, are able to distinguish the gp52 of C3H MMTV from the gp52's of GR and RIII MMTV.     

5-Fluoro-2'-deoxyuridine incorporation in L1210 DNA

We have employed cesium sulfate density gradient centrifugation to separate RNA and DNA of L1210 cells labeled with [3H]fluorodeoxyuridine. We have analyzed nucleotide and nucleoside digests of purified DNA from the [3H]fluorodeoxyuridine-labeled cells and demonstrate by reverse phase and anion exchange high pressure liquid chromatography the presence of tritium radioactivity co-migrating with fluorodeoxyuridine 5'-monophosphate or fluorodeoxyuridine. These observations demonstrate the internucleotide incorporation of fluorodeoxyuridine in DNA and suggest a new mechanism of action for this cytotoxic and mutagenic agent.     

Solubilization, Characterization, and Partial Purification of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-, Quisqualate-, Kainate-Sensitive l-Glutamate Binding Sites from Porcine Brain Synaptic Junctions

L-[3H]Glutamate binding sites were solubilized from porcine brain synaptic junctions by Triton X-114 in the presence of KCl. The solubilized binding sites bound L-[3H]glutamate reversibly with KD and Bmax values of 1.48 +/- 0.18 microM and 178.2 +/- 15.9 pmol/mg of protein, respectively. These binding sites appeared to be integral membrane glycoproteins, with sugar moieties recognized by wheat germ agglutinin. A 49.3-fold purification of these binding sites was achieved by Triton X-114 solubilization, anion-exchange chromatography, and affinity chromatography using wheat germ agglutinin-Sepharose. The apparent molecular mass of the partially purified binding sites was 620 +/- 50 kDa. L-[3H]Glutamate bound to the solubilized preparation could be effectively displaced by agonists of non-N-methyl-D-aspartate (NMDA) L-glutamate receptors but not by NMDA or alpha-amino-4-phosphonobutyrate. The rank order for the competitive ligands in displacing L-[3H]glutamate was: quisqualate greater than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid greater than L-glutamate greater than kainate.     

The oligosaccharide component of alpha 1-adrenergic receptors from BC3H1 and DDT1 muscle cells. Studies with glycosidases and photoaffinity labelling of intact cells.

In this study, we clarify the structural aspects of the oligosaccharides associated with the alpha 1-adrenergic receptor in two muscle cell lines. Photoaffinity labelling of intact BC3H1 or DDT1 muscle cells with 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline ([125I]azidoprazosin) followed by SDS/polyacrylamide-gel electrophoresis (PAGE) and autoradiography revealed specifically labelled proteins of molecular mass = 87,000 and 81,000, respectively. Treatment of photoaffinity-labelled receptors in DDT1 cells with 33 u. of endoglycosidase F/ml for 24 h resulted in the loss of the 81 kDa receptor and the appearance of a 52.5 kDa protein. When lower concentrations of glycosidase or shorter incubation times were used, the 81 kDa receptor was converted to a 66 kDa protein. Treatment of the photoaffinity-labelled BC3H1 receptor with endoglycosidase F resulted in the appearance of a 50.5 kDa protein. Neither alpha-mannosidase nor endoglycosidase H had an effect on the photoaffinity labelling patterns of the receptor from the two cell types. alpha 1-Adrenergic receptors, solubilized from membranes prepared from BC3H1 and DDT1 cells, bound to wheat germ agglutinin-Sepharose and were displaced by N-acetylglucosamine. Taken together, these results indicate that alpha 1-adrenergic receptors in BC3H1 and DDT1 cells contain complex, but not high, mannose oligosaccharide chains; differences in the composition or number of chains partially accounts for the different molecular mass of the receptor in the two cell lines. The results further indicate that the oligosaccharide chains contribute substantially to the apparent molecular mass of alpha 1-adrenergic receptors, as detected by SDS/PAGE, and that the protein backbone of these receptors is likely to be approximately 50 kDa.     

Photoaffinity-labelling of the calcium-channel-associated 1,4-dihydropyridine and phenylalkylamine receptor in guinea-pig hippocampus. A 195 kDa polypeptide carries both drug receptors and has similarities to the alpha 1 subunit of the purified skeletal-muscle calcium channel.

This study identifies calcium-antagonist-receptor-carrying polypeptides of calcium channels in guinea-pig hippocampus membranes. The arylazide ligands (-)-[3H]azidopine and [N-methyl-3H]LU49888 [(-)-5-[(3-azidophenethyl) [N-methyl-3H]methylamino]-2-(3,4,5-trimethoxyphenyl-2- isopropylvaleronitrile] were used to selectively label 1,4-dihydropyridine and phenylalkylamine receptors respectively. In the absence of u.v. light, both ligands reversibly bound to a single class of high-affinity receptors with a calcium-channel-typical pharmacological profile. [N-methyl-3H]LU49888 bound to the extent of 849 +/- 188 fmol/mg of protein (mean +/- S.D., n = 3) with a dissociation constant (Kd) of 1.4 +/- 0.3 nM. Under identical assay conditions (-)-[3H]azidopine labelled to the extent of 562 +/- 132 fmol/mg of protein with a Kd of 0.096 +/- 0.024 nM. After u.v. irradiation of the [N-methyl-3H]LU49888- and (-)-[3H]azidopine-labelled membranes, both photo-affinity probes were found to be incorporated specifically into a 190-195 kDa band as shown by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE). Photoincorporation occurred with a protection profile identical with that produced by reversible binding-inhibition. [N-methyl-3H]LU49888, but not (-)-[3H]-azidopine, specifically labelled an additional 265 kDa band. Both photolabelled bands had an identical electrophoretic mobility on SDS/PAGE, irrespective of pretreatment either with 10 mM-N-ethylmaleimide or 10 mM-dithiothreitol. The electrophoretic properties of the 195 kDa polypeptide and the lability of receptor-incorporated (-)-[3H]azidopine to nucleophilic agents resemble those of the previously described drug-receptor-carrying alpha 1 subunit of the purified skeletal-muscle calcium channel. The data suggest that this polypeptide carries both the high-affinity 1,4-dihydropyridine as well as the phenylalkylamine receptor of neuronal calcium channels in guinea-pig hippocampus and is a component of the L-type calcium channel.     

Cytotoxic activity of platinum (II) complexes with tri-n-butylphosphine. Crystal structure of the dinuclear hydrazine-bridged complex, cis,cis-[PtCl(PBu3n)2(mu-N2H4)PtCl(PBu3n) 2] (ClO4)2.2CHCl3.

A series of platinum(II) tri-n-butylphosphine complexes having the formulas cis-[PtCl2L2], NEt4[PtCl3L], [PtCl(en)L]Cl, [Pt(en)L2](ClO4)2, sym-trans-[Pt2Cl4L2], [Pt2Cl2L4](ClO4)2, trans,trans-[PtCl2L(mu-N2H4)PtCl2L] trans,trans-[PtCl2L(mu-en)PtCl2L], and cis,cis-[PtClL2(mu-N2H4)PtClL2](ClO4)2 (L = tri-n-butylphosphine; en = ethylenediamine) have been synthesized and their cytotoxic activity in vitro and in vivo has been studied. The solution behavior of the novel dinuclear diamine-bridged platinum(II) complexes has been investigated by means of UV and 31P NMR spectroscopy. For the ionic hydrazine compound cis,cis-[PtClL2(mu-N2H4)PtClL2](ClO4)2, an x-ray structure determination is reported. Crystal data: space group P2(1)/a, a = 17.803(1), b = 18.888(3), c = 12.506(3) A, beta = 107.97(2) degrees, Z = 2, R = 0.052, RW = 0.058. The platinum coordination is approximately square-planar, with the bond lengths Pt-Cl = 2.358(5), Pt-N = 2.15(1), Pt-P(trans to Cl) = 2.260(5), and Pt-P(trans to N) = 2.262(6) A. All investigated compounds were cytotoxic in vitro against L1210 cells and showed no cross-resistance to cisplatin. On the other hand, no antitumor activity was observed vs L1210 leucemia in DBA2 mice.     

Purification to apparent homogeneity and partial amino acid sequence of rat liver O6-alkylguanine-DNA-alkyltransferase.

O6-alkylguanine-DNA-alkyltransferase (ATase) activity was increased in rat liver from 80 to 320 fmoles/mg total protein 48 h after administration of 2-acetylaminofluorene at 60 mg/kg body weight. This tissue was used as a source of ATase which was purified by ammonium sulphate precipitation and DNA-cellulose, molecular exclusion and ion exchange chromatography (IEC). IEC purified material showed a major 24 kDa band after polyacrylamide gel electrophoresis (PAGE) with silver staining. Fluorography of purified ATase following incubation with [3H]-methylated substrate DNA and PAGE showed a single band at 24 kDa suggesting that, as with bacterial ATases, the protein itself accepts the alkyl group from O6-alkylguanine in substrate DNA during the repair reaction. Further purification of the protein using reverse phase HPLC resulted in a single peak representing approximately 125,000 fold purification. This was subjected to amino-terminal sequencing and it was found that the protein was blocked at the amino-terminal end: it was cleaved using trypsin or cyanogen bromide and the amino acid sequence of several reverse phase HPLC purified fragments was determined.     

In vivo phosphorylation of distinct domains of the 70-kilodalton neurofilament subunit involves different protein kinases

A combination of in vivo and in vitro approaches were used to characterize phosphorylation sites on the 70,000-kilodalton (kDa) subunit of neurofilaments (NF-L) and to identify the protein kinases that are likely to mediate these modifications in vivo. Neurofilament proteins in a single class of neurons, the retinal ganglion cells, were pulse-labeled in vivo by injecting mice intravitreously with [32P]orthophosphate. Radiolabeled neurofilaments were isolated after they had advanced along optic axons, and the individual subunits were separated on sodium dodecyl sulfate-polyacrylamide gels. Two-dimensional alpha-chymotryptic phosphopeptide map analysis of NF-L revealed three phosphorylation sites: an intensely labeled peptide (L-1) and two less intensely labeled peptides (L-2 and L-3). The alpha-chymotryptic peptide L-1 was identified as the 11-kDa segment containing the C terminus of NF-L. The ability of these peptides to serve as substrates for specific protein kinases were examined by incubating neurofilament preparations with [gamma-32P]ATP in the presence of purified cAMP-dependent protein kinase or appropriate activators and/or inhibitors of endogenous cytoskeleton-associated protein kinases. The heparin-sensitive, calcium- and cyclic nucleotide-independent kinase associated with the cytoskeleton selectively phosphorylated L-1 and L-3 but had little, if any, activity toward L-2. When this kinase was inhibited with heparin, cAMP addition to the neurofilament preparation stimulated the phosphorylation of L-2, and addition of the purified catalytic subunit of cAMP-dependent protein kinase induced intense labeling of L-2. At higher labeling efficiencies, the exogenous kinase also phosphorylated L-3 and several sites at which labeling was not detected in vivo; however, L-1 was not a substrate. Calcium and calmodulin added to neurofilament preparations in the presence of heparin modestly stimulated the phosphorylation of L-1 and L-3, but not L-2, and the stimulation was reversed by trifluoperazine. The selective phosphorylation of different polypeptide domains on NF-L by second messenger-dependent and -independent kinases suggests multiple functions for phosphate groups on this protein.     

Comparative characteristics of the antitumor and immunodepressive activity of carminomycin on the L-1210 experimental model]

The antitumor activity of carminomycin was estimated by the number of lymphoma colonies formed in the spleen of DBA/2 mice on their inoculation with the bone marrow cells from mice with transplantable leukemia L-1210. The immunodepressive properties of carminomycin were determined by the number of the antibody forming cells in the spleen of CBA and DBA/2 mice with leukemia L-1210 after immunization with sheep red blood cells. It was found that in a single dose of 1.5 mg/kg carminomycin inhibited the lymphoma colonies by 50 per cent. The maximum immunodepressive effect was observed when carminomycin was used in a single dose of 1.5 mg/kg 48 hours after the antigen stimulation. In this case the number of the antibody forming cells in DBA/2 mice with leukemia L-1210 was lower than that in CBA mice without leukemia.     

Rapid purification of two lipocortin-like proteins from mice lung

We have purified two proteins (40 kDa and 36 kDa) from mice lung by the method of calcium-precipitation/EGTA solubilization and then a separation on a high anion exchanger column (Mono Q HR 5/5. Pharmacia) with a gradient of NaCl. The two proteins were strong inhibitors of phospholipase A2 as assessed in vitro with porcine pancreatic phospholipase A2 and [3H]-oleic acid labeled E. Coli membranes as substrate. The 40 kDa protein had a pI of 5.8 and was found to be immunologically related to human recombinant lipocortin I. The 36 kDa protein had a pI of 4.7 and cross-reacted with a polyclonal antibody raised against a 32 kDa human lipocortin-like protein described in human blood mononuclear cells. We report here a rapid purification of two distinct lipocortin-like proteins from mice lung.     

Identification of cell membrane proteins that bind visna virus.

Visna virus infects cells of ovine origin by attaching to a cell surface receptor via its envelope glycoprotein. The identity of the visna virus receptor is not known. To identify the molecule responsible for binding the virus to target cells, virus overlay protein blot assays were used to examine the molecular weights of cell surface molecules which bind purified virus. Molecules on the surface of goat synovial membrane (GSM) cells and sheep choroid plexus (SCP) cells of approximately 15, 30, and 50 kDa bound to visna virus. The binding of visna virus to these proteins was reduced by preincubating virus with neutralizing antibodies. 125I-labeled cell membrane preparations of GSM and SCP cells were used to affinity purify these virus-binding proteins. These proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had molecular masses of 15, 30, and 50 kDa. Antibodies to the 50-kDa protein bound to the surface of both live SCP and GSM cells in immunofluorescence assays. In addition, antibodies to the 50-kDa protein blocked the binding of [35S]methionine-labeled visna virus to SCP cells in culture. Antibodies raised against the 15- and 30-kDa proteins did not block virus binding to cells. The blocking activity of antibody of the 50-kDa protein provided data that this protein is the molecule which visna virus recognizes and binds to on the surface of target cells.     

Purification and properties of extracellular mutacin, a bacteriocin from Streptococcus sobrinus.

Mutacin MT6223, a cell-free bacteriocin produced by Streptococcus sobrinus MT6223, was purified by ammonium sulphate precipitation, chromatofocusing with PBE 94 and column chromatography on SP Sephadex C-25. The specific activity of the purified mutacin was increased 1950-fold with a recovery of 9.7%. The molecular mass of the purified mutacin preparation was estimated to be 6.5 kDa. The mutacin activity was stable from pH 2-7, and was resistant to treatment at 100 degrees C for 20 min. It was inactivated by papain or ficin digestion, and was partially inhibited by alpha-chymotrypsin. The mutacin was found to be active against strains of serotypes c, e and f of Streptococcus mutans and the addition of purified mutacin MT6223 to growing cells of S. mutans MT8148 resulted in a rapid inhibition of incorporation of [3H]thymidine, [3H]uracil or L-[3H]glutamic acid into DNA, RNA or protein, respectively. Specific pathogen-free Fischer rats fed diet 2000 and infected with S. mutans MT8148R showed significantly fewer caries and lower plaque scores when mutacin was administered through drinking water. The present study demonstrates that mutacin MT6223 inhibited the growth of mutans streptococci. Thus, mutacin MT6223 may be a candidate for use in dental caries prevention.     

Characterization of a peripheral-type benzodiazepine-binding site in the mitochondria of Chinese hamster ovary cells

The isoquinoline carboxamide derivative [3H]PK11195, a ligand for the peripheral-type benzodiazepine (BZD) receptor, binds to Chinese hamster ovary (CHO) cell mitochondria in a specific and saturable manner. Scatchard analysis showed the presence of a single-binding site with an apparent dissociation constant (Kd) of 12.0 +/- 1.0 nM and a maximal binding capacity of 23.0 +/- 2.0 pmol/mg protein. The pharmacological characterization of this CHO BZD-binding site, based on the displacement of [3H]PK11195 by several drugs of known binding specificity, indicated that it is of the peripheral-type. The photoaffinity probe [3H]PK14105, a nitrophenyl derivative of [3H]PK11195, specifically labeled a 17 kDa CHO mitochondrial protein. This 17 kDa protein was purified from digitonin-solubilized mitochondria by gel-filtration chromatography and two reverse-phase HPLC steps. The purified material migrated as a single band on silver stained or autoradiographed SDS-polyacrylamide gels, and had an amino acid composition corresponding to a 17 kDa protein rich in Leu, Val, Ala, Gly, and Pro. Analysis of the amino-terminal sequence of the purified 17 kDa protein revealed a blocked amino-terminus.     

Further studies on a novel class of genetic variants of the L1210 cell with increased folate analogue transport inward. Transport properties of a new variant, evidence for increased levels of a specific transport protein, and its partial characterization following affinity labeling

Studies are reported on the characterization of a new isolate within a novel class of variants of the L1210 cell exhibiting markedly increased transport inward of folate analogues. This variant (L1210/R83), which was selected in the presence of the antifolate metoprine, exhibited a 40-fold increase in [3H]aminopterin influx compared to parental cells and a modest (4-5-fold) increase in [3H]aminopterin efflux. The increase in influx was associated with a comparable increase in influx Vmax for the one-carbon, reduced folate transport system and the same increase in the amount of specific binding of [3H]aminopterin on the cell surface. Values for influx Km for [3H]aminopterin and specificity for various folate structures were unchanged. The alteration in influx Vmax and more rapid efflux accounted for the different level of intracellular exchangeable level of drug at steady state in this variant compared with parental L1210 cells. Otherwise, membrane potential was unchanged. The N-hydroxysuccinimide ester of [3H]aminopterin was used to covalently label the specific binding protein for folate compounds in the plasma membrane of variant and parental L1210 cells. Incorporation of label into this protein was stable under a variety of conditions and accounted for 97 and 52% of total cellular labeling, respectively, for membrane derived from R83 and parental L1210 cells at a reagent concentration of 20 nM. Specific affinity labeling on the surface of parental and variant cells was decreased in the presence of aminopterin, methotrexate, or 5-formyltetrahydrofolate, but not in the presence of folic acid. Also, [3H]aminopterin influx in these cells was inhibited by the N-hydroxysuccinimide ester of aminopterin or methotrexate, but not the N-hydroxysuccinimide ester of folic acid. These findings, in addition to the increased affinity labeling of this variant, which corresponds to the increase in influx of [3H] aminopterin also seen, appears to identify the affinity labeled protein as a component of the "classical" one-carbon, reduced folate transport system in these cells. The affinity labeled protein from each cell type was solubilized in sodium dodecyl sulfate or extracted in detergent in the presence of proteinase inhibitors and was found to elute from Sephacryl S-300 and migrate during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single peak of Mr = 45,000-48,000. Recovery of labeled binding protein in these fractions from R83 variant cells was approximately 40 times greater than that from parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)