Synthesis and properties of heterobifunctional photoaffinity reagents for the identification and isolation of receptors

A method employing isotopically- and photoaffinity-labeled probes and polyclonal and monoclonal antibody to the probes for the identification, isolation and recovery of protein receptors is described. Antibody was raised against N-(3-(p-azido-m-($\sp{125}$I) -iodophenyl)) propionate (AIPP) coupled to and photolyzed to BSA. The antibodies specifically bound AIPP-derivatized proteins. An isolation system was developed utilizing this probe and two antigenically identical reversible analogues. N-(3-((p-azido-m-($\sp{125}$I) -iodo-phenyl)propionyl)amidoethyl-1,3-dithiopropionyl) succinimide (Reversible $\sp{125}$I-AIPPS) reacts with primary amines and N-(((3-p-azido-m-($\sp{125}$I) -iodophenyl)propionyl)amidoethyl)dithiopyridine ($\sp{125}$I-AIPP-PDA) reacts with reduced thiols. The applicability of the system was established by derivatizing known ligands (Transferrin and Interferon-alpha) with one of the probes. The ligand-probe was then allowed to interact with its receptor by incubation with SS5 lymphoma cells and cross-linked by photolysis at 300 nm. The photolyzed ligand/probe/receptor preparation was then recovered with AIPP antibody. Utilization of N-(3-((p-azido-m-($\sp{125}$I) -iodo-phenyl-propionyl)-amidoethyl-1,3-dithiopropionyl) succinimide (Reversible $\sp{125}$I-AIPPS) allowed the components of the photolyzed complex to be separated by treatment with 2-mercaptoethanol in the SDS-PAGE solubilization buffer. Ligand and receptor labeling were then assessed by Coomassie staining and autoradiography. Results of receptor assays suggest that $\sp{125}$I-AIPP was, indeed, transferred to moieties that represent the receptors for both Transferrin and Interferon-alpha. ^

Cytochrome P450 in mammalian brain: Purification, anatomical distribution and regulation of the cytochrome P450 monooxygenase system in rat brain

The cytochrome P450 (P450) monooxygenase system plays a major role in metabolizing a wide variety of xenobiotic as well as endogenous compounds. In performing this function, it serves to protect the body from foreign substances. However, in a number of cases, P450 activates procarcinogens to cause harm. In most animals, the highest level of activity is found in the liver. Virtually all tissues demonstrate P450 activity, though, and the role of the P450 monooxygenase system in these other organs is not well understood. In this project I have studied the P450 system in rat brain; purifying NADPH-cytochrome P450 reductase (reductase) from that tissue. In addition, I have examined the distribution and regulation of expression of reductase and P450 in various anatomical regions of the rat brain.^ NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by SDS-PAGE and Western blot techniques. Kinetic studies utilizing cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P4501A1 as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. These results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.^ Since the brain is not a homogeneous organ, dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monooxygenase system in brain is important in elucidating its function in that organ. Related to this is the regulation of P450 expression in brain. In order to study these issues female rats--both ovariectomized and not--were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and reductase determined using polymerase chain reaction. Results of this study indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR has allowed the determination of factors affecting the expression of message for these enzymes. ^

Protein quaternary structure determination using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and chemical crosslinking

A means of analyzing protein quaternary structure using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI MS) and chemical crosslinking was evaluated. Proteins of known oligomeric structure, as well as monomeric proteins, were analyzed to evaluate the method. The quaternary structure of proteins of unknown or uncertain structure was investigated using this technique. The stoichiometry of recombinant E. coli carbamoyl phosphate synthetase and recombinant human farnesyl protein transferase were determined to be heterodimers using glutaraldehyde crosslinking, agreeing with the stoichiometry found for the wild type proteins. The stoichiometry of the gamma subunit of E. coli DNA polymerase III holoenzyme was determined in solution without the presence of other subunits to be a homotetramer using glutaraldehyde crosslinking and MALDI MS analysis. Chi and psi subunits of E. coli DNA polymerase III subunits appeared to form a heterodimer when crosslinked with heterobifunctional photoreactive crosslinkers.^ Comparison of relative % peak areas obtained from MALDI MS analysis of crosslinked proteins and densitometric scanning of silver stained sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels showed excellent qualitative agreement for the two techniques, but the quantitative analyses differed, sometimes significantly. This difference in quantitation could be due to SDS-PAGE conditions (differential staining, loss of sample) or to MALDI MS conditions (differences in ionization and/or detection). Investigation of pre-purified crosslinked monomers and dimers recombined in a specific ratio revealed the presence of mass discrimination in the MALDI MS process. The calculation of mass discrimination for two different MALDI time-of-flight instruments showed the loss of a factor of approximately 2.6 in relative peak area as the m/z value doubles over the m/z range from 30,000 to 145,000 daltons.^ Indirect symmetry was determined for tetramers using glutaraldehyde crosslinking with MALDI MS analysis. Mathematical modelling and simple graphing allowed the determination of the symmetry for several tetramers known to possess isologous D2 symmetry. These methods also distinguished tetramers that did not fit D2 symmetry such as apo-avidin. The gamma tetramer of E. coli DNA polymerase III appears to have isologous D2 symmetry. ^

Cytochrome P450 2D18 in the mammalian brain: Recombinant expression, purification, and characterization of xenobiotic and endobiotic metabolism

The cytochromes P450 (P450) comprise a superfamily of hemoproteins that function in concert with NADPH-cytochrome P450 reductase (P450-reductase) to metabolize both endogenous and exogenous compounds. Many pharmacological agents undergo phase I metabolism by this P450 and P450-reductase monooxygenase system. Phase I metabolism ensures that these highly hydrophobic xenobiotics are made more hydrophilic, and hence easier to extrude from the body. While the majority of phase I metabolism occurs in the liver, metabolism in extrahepatic organ-systems like the intestine, kidney, and brain can have important roles in drug metabolism and/or efficacy. ^ While P450-mediated phase I metabolism has been well studied, investigators have only recently begun to elucidate what physiological roles P450 may have. One way to approach this question is to study P450s that are highly or specifically expressed in extrahepatic tissues. In this project I have studied the role of a recently cloned P450 family member, P450 2D18, that was previously shown to be expressed in the rat brain and kidney, but not in the liver. To this end, I have used the baculovirus expression system to over-express recombinant P450 2D18 and purified the functional enzyme using nickel and hydroxylapatite chromatography. SDS-PAGE analysis indicated that the enzyme was purified to electrophoretic homogeneity and Western analysis showed cross-reactivity with rabbit anti-human P450 2D6. Carbon monoxide difference spectra indicated that the purified protein contained no denatured P450 enzyme; this allowed for further characterization of the substrates and metabolites formed by P450 2D18-mediated metabolism. ^ Because P450 2D18 is expressed in brain, we characterized the activity toward several psychoactive drugs including the antidepressants imipramine and desipramine, and the anti-psychotic drugs chlorpromazine and haloperidol. P450 2D18 preferentially catalyzed the N-demethylation of imipramine, desipramine, and chlorpromazine. This is interesting given the fact that other P450 isoforms form multiple metabolites from such compounds. This limited metabolic profile might suggest that P450 2D18 has some unique function, or perhaps a role in endobiotic metabolism. ^ Further analysis of possible endogenous substrates for P450 2D18 led to the identification of dopamine and arachidonic acid as substrates. It was shown that P450 2D18 catalyzes the oxidation of dopamine to aminochrome, and that the enzyme binds dopamine with an apparent KS value of 678 μM, a value well within reported dopamine concentration in brain dopaminergic systems. Further, it was shown that P450 2D18 binds arachidonic acid with an apparent KS value of 148 μM, and catalyzes both the ω-hydroxylation and epoxygenation of arachidonic acid to metabolites that have been shown to have vasoactive properties in brain, kidney, and heart tissues. These data provide clues for endogenous roles of P450 within the brain, and possible involvement in the pathogenesis of Parkinson's disease. ^

MOUSE MAMMARY TUMOR VIRUS EPITOPES EXPRESSED ON TUMOR SPECIFIC TRANSPLANTATION ANTIGENS OF CHEMICALLY INDUCED C3H/HEJ SARCOMAS (TSTA, FIBROSARCOMA, CARCINOGENESIS, MMTV, METHYLCHOLANTHRONE)

This dissertation addressed the hypothesis that the unique tumor specific transplantation antigens (TSTA) of chemically induced sarcomas express epitopes encoded by endogenous viral genes. TSTA from two 3-methylcholanthrene-induced, C3H/HeJ fibrosarcomas (MCA-F and MCA-D) were serologically assessed for viral epitopes in an enzyme-linked immunospecific assay (ELISA) and by immunoaffinity chromatography. Initial evidence with an anti-TSTA antiserum suggested that TSTA were associated with mouse mammary tumor virus (MMTV) peptides, but not peptides from murine leukemia virus (MuLV). TSTA extracted from MCA-F, was assessed with specific anti viral antibodies at three levels of purification for its association with MuLV peptides (gp 70 and p 15E) and MMTV peptides (gp52, gp36 and p27). The results demonstrate that purified preparations enriched for TSTA activity are devoid of MuLV epitopes, but enriched for a subset of MMTV epitopes. Immunoaffinity supports constructed with anti-MMTV antibodies retained TSTA from partially purified MCA-F or MCA-D extracts. Immunoaffinity chromatography with antibodies against individual MMTV peptides demonstrated that the MCA-F TSTA was specifically retained by anti-gp36 and anti-p27 supports, but not by anti-gp52 supports nor a support made with bovine serum albumin. Analysis of the affinity purified TSTA preparations by HPGPC and SDS-PAGE revealed only a few components. Application of the anti-gp36 and anti-p27 retained materials to HPGPC and subsequent in vivo analysis demonstrated that the TSTA migrated in a low and a high molecular weight region. These results suggest that TSTA specificity in C3H/HeJ mice, results from MMTV recombinant proteins. ^

CHARACTERIZATION OF HUMAN ACID ALPHA-GLUCOSIDASE

The goal of this study was to investigate the properties of human acid (alpha)-glucosidase with respect to: (i) the molecular heterogeneity of the enzyme and (ii) the synthesis, post-translational modification, and transport of acid (alpha)-glucosidase in human fibroblasts.^ The initial phase of these investigations involved the purification of acid (alpha)-glucosidase from the human liver. Human hepatic acid (alpha)-glucosidase was characterized by isoelectric focusing and native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four distinct charge forms of hepatic acid (alpha)-glucosidase were separated by chromatofocusing and characterized individually. Charge heterogeneity was demonstrated to result from differences in the polypeptide components of each charge form.^ The second aspect of this research focused on the biosynthesis and the intracellular processing and transport of acid (alpha)-glucosidase in human fibroblasts. These experiments were accomplished by immune precipitation of the biosynthetic intermediates of acid (alpha)-glucosidase from radioactively labeled fibroblasts with polyclonal and monoclonal antibodies raised against human hepatic acid (alpha)-glucosidase. The immune precipitated biosynthetic forms of acid (alpha)-glucosidase were analyzed by SDS-PAGE and autoradiography. The pulse-chase experiments demonstrated the existence of several transient, high molecular weight precursors of acid (alpha)-glucosidase. These precursors were demonstrated to be intermediates of acid (alpha)-glucosidase at different stages of transport and processing in the Golgi apparatus. Other experiments were performed to examine the role of co-translational glycosylation of acid (alpha)-glucosidase in the transport and processing of precursors of this enzyme.^ A specific immunological assay for detecting acid (alpha)-glucosidase was developed using the monoclonal antibodies described above. This method was modified to increase the sensitivity of the assay by utilization of the biotin-avidin amplification system. This method was demonstrated to be more sensitive for detecting human acid (alpha)-glucosidase than the currently used biochemical assay for acid (alpha)-glucosidase activity. It was also demonstrated that the biotin-avidin immunoassay could discriminate between normal and acid (alpha)-glucosidase deficient fibroblasts, thus providing an alternative approach to detecting this inborn error in metabolism. (Abstract shortened with permission of author.) ^

Regulation of sex hormone dependent growth by a serum borne inhibitor

This thesis project focused on understanding the basic process controlling cell proliferation in sex-steroid hormone dependent cancers. The availability of inculture models using cloned cell lines offers the greatest advantage for studying the control of this event. Incubation of cloned sex-hormone sensitive cells in medium containing increasing concentrations of sex-hormone stripped serum, results in a dose dependent growth inhibition; this inhibition is reversed by the addition of physiological concentrations of steroid hormones. The mechanisms explaining this phenomenon are not yet fully understood, but different theories propose the existence in serum of a sex hormone binding protein with growth inhibitory properties. We were able to identify a protein that specifically binds sex hormones in rat and horse serum with affinities 10-fold lower to the ones observed with the classic sex-hormone binding globulin (SHBG) in humans. Purification of this protein on a large scale Lowed a more detailed analysis. The putative sex-hormone binding protein has an apparent molecular weight of 386 KDa. SDS-PAGE with commassie staining of the purified product, displayed a pattern non-characteristic of SMG, but all bands cross-reacted with a commercial anti-SMG antibody in western analysis. Titrations of the purified product on cell proliferation assays using sex-hormone dependent lines, resulted in a dose dependent growth inhibition. This inhibition was reversed by the addition of sex hormones. Our results indicate that we have identified and purified a sex-hormone binding protein in serum with characteristics similar to SHBG and with cell growth inhibitory properties. ^

Contribution of transmembrane and cytoplasmic domains to membrane protein association

Membrane proteins are critical to every aspect of cell physiology, with their association mediating important biological functions. The transmembrane and cytoplasmic domains are known to be important for their association. In order to characterize their role in detail, we have applied different biophysical techniques in detergent micelles to two model systems. The first study involves FcRγ, a single transmembrane domain protein existing as a disulfide linked homodimer. We investigated the role of a conserved transmembrane polar residue and the cytoplasmic tail in FcRγ homo-interactions. Our results by various biophysical techniques including SDS-PAGE, circular dichroism and sedimentation equilibrium in detergent micelles indicate importance of both the transmembrane polar residue and cytoplasmic tail in maintaining proper conformation for FcRγ homo-interactions. A contrasting second study was on L-selectin, another single transmembrane domain protein with a large extracellular domain and a short cytoplasmic tail. Previous cross-linking experiments indicate its possible dimerization. However, the purified fragment of L-selectin and corresponding mutants did not dimerize when analyzed by TOXCAT assay, sedimentation equilibrium and fluorescence resonance energy transfer. It was likely that the presence of juxtamembrane positively charged residues led to decreased migrational rates in SDS PAGE. In conclusion, complementary biophysical techniques should be used with care when studying membrane protein association in detergent micelles. As an extension to our study on L-selectin, we also investigated its interaction with Calmodulin (CaM) in detergent micelles. CaM was found to interact with different detergents. We applied fluorescence and NMR spectroscopy to characterize the interaction of both the apo and Ca 2+ bound form of CaM, with commonly used detergents, below and above their respective critical micelle concentrations. The interaction of apo-CaM with detergents was found to vary with the nature of the detergent head group, whereas Ca2+-CaM interacted with individual detergent molecules irrespective of the nature of their head group. NMR titration experiments of CaM with detergents indicated involvement of specific residues from the N-lobe, linker and C-lobe of CaM. ^

DIAGNOSTIC ANTIGENS FOR SCHISTOSOMIASIS MANSONI: PRODUCTION, CHARACTERIZATION AND PURIFICATION OF MONOCLONAL ANTIBODIES, AND AFFINITY PURIFICATION OF ANTIGENS

Attempts have been made in this dissertation to develop a purified antigen with high sensitivity and specificity for diagnosis of Schistosoma mansoni (Sm) infection by using the hybridoma technique.^ Spleen cells, obtained from mice immunized by infection with Sm and boosted by cercarial antigens, or by injection of circulating antigen (CA) in serum from infected mice, were fused with Sp2/0 myeloma cells. The active infection resulted a higher number of hybridomas (100%) than by CA (20%), and higher levels of antibody reactivity as measured by ELISA.^ The IgM and IgG monoclonal antibodies (MCAbs) were purified respectively by gel filtration, DE 52 ion exchange column and proteinase A affinity column. The cercarial and egg antigens were purified by affinity chromatography through MCAb/affi-gel column. The reactivity of the purified antigens were then monitored by ELISA, SDS-PAGE silver stain and EITB.^ The respective MCAbs recognized varying antigenic determinants (AD) present in adult, cercaria and egg stages. By EITB the MCAbs IgM and IgG, when reacted with nine antigens from the various stages, revealed identical bands, suggesting that the two MCAb classes originated from identical AD. By ELISA and COPT, the MCAbs from thirteen cell lines gave same results. But by CHR, two MCAbs showed negative results while eleven other MCAbs showed strong positive. It is assumed that the AD in the immunogen that ilicited the MCAbs were immunochemically closely related.^ One egg purified by immunoaffinity indicated that the epitopes recognized by MCAb were present on four antigenic components with molecular weights (Mr) of approximately 19, 25, 60 and >224 kd, respectively. By EITB the Mr 19 doublet appeared to be species specific; the Mr 25 kd genus specific. They reacted with mouse serum from 13-16 weeks after infection. In monkey serum Mr 19 doublet appeared 8-10 weeks after infection and disappeared at 8-12 weeks after Droncit treatment, paralleled to the disappearance of fecal egg. The Mr 60 and >224 kd bands were also demonstrated with S. japonicum, S. haematobium and Trichinella spiralis infection sera and may be the cause of cross-reaction in conventional serological test. ^

EVIDENCE OF HUMAN ENDOGENOUS RETROVIRUS K INVOLVEMENT IN HUMAN CANCER

Many lines of clinical and experimental evidence indicate a viral role in carcinogenesis (1-6). Our access to patient plasma, serum, and tissue samples from invasive breast cancer (N=19), ductal carcinoma in situ (N=13), malignant ovarian cancer (N=12), and benign ovarian tumors (N=9), via IRB-approved and informed consent protocols through M.D. Anderson Cancer Center, as well as normal donor plasmas purchased from Gulf Coast Regional Blood Center (N=6), has allowed us to survey primary patient blood and tissue samples, healthy donor blood from the general population, as well as commercially available human cell lines for the presence of human endogenous retrovirus K (HERV-K) Env viral RNA (vRNA), protein, and viral particles. We hypothesize that HERV-K proteins are tumor-associated antigens and as such can be profiled and targeted in patients for diagnostic and therapeutic purposes. To test this hypothesis, we employed isopycnic ultracentrifugation, a microplate-based reverse transcriptase enzyme activity assay, reverse transcription – polymerase chain reaction (RT-PCR), cDNA sequencing, SDS-PAGE and western blotting, immunofluorescent staining, confocal microscopy, and transmission electron microscopy to evaluate v HERV-K activation in cancer. Data from large numbers of patients tested by reverse transcriptase activity assay were analyzed statistically by t-test to determine the potential use of this assay as a diagnostic tool for cancer. Significant reverse transcriptase enzyme activity was detected in 75% of ovarian cancer patients, 53.8% of ductal carcinoma in situ patient, and 42.1% of invasive breast cancer patient samples. Only 11.1% of benign ovarian patient and 16.7% of normal donor samples tested positive. HERV-K Env vRNA, or Env SU were detected in the majority of cancer types screened, as demonstrated by the results shown herein, and were largely absent in normal controls. These findings support our hypothesis that the presence of HERV-K in patient blood circulation is an indicator of cancer or pre-malignancy in vivo, that the presence of HERV-K Env on tumor cell surfaces is indicative of malignant phenotype, and that HERV-K Env is a tumor-associated antigen useful not only as a diagnostic screening tool to predict patient disease status, but also as an exploitable therapeutic target for various novel antibody-based immunotherapies.

The role of suppressor factors in the regulation of immune responses by ultraviolet radiation -induced suppressor T lymphocytes

The purpose of these studies was to determine the role of suppressor factors (TsF) in the regulation of immune responses by ultraviolet radiation-induced suppressor T lymphocytes (Ts). The Ts were induced following epicutaneous sensitization with contact allergens to an unirradiated site on mice irradiated five days earlier with 40 kJ/m$\sp2$ UVB (280-320 nm) radiation. The spleens of such mice contain afferent, hapten-specific, Thy-1$\sp+$, Lyt-1$\sp+$,2$\sp-$ Ts that suppress in vivo contact hypersensitivity (CHS) and antibody responses and the in vitro generation of cytotoxic T lymphocytes (CTL). Four approaches were used to determine the role of TsF. First, lysates produced from sonically-disrupted Ts were injected i.v. into normal animals; they inhibited CHS in vivo in a nonspecific manner. The lysates suppressed the induction and elicitation of CHS, and they inhibited the in vitro generation of CTL. Lysates prepared from splenocytes obtained from unirradiated mice or UV-irradiated, unsensitized mice failed to inhibit either response. Second, supernatants from cultures containing Ts, normal syngeneic responder lymphocytes, and hapten-modified stimulator cells were injected i.v. into normal recipients. They inhibited the induction of CHS and did so in a hapten-specific manner. Cellular and kinetic requirements were observed for the generation of suppressive activity. Splenocytes from mice treated with Ts supernatants suppressed CHS when transferred into normal animals. The supernatants also suppressed the in vitro generation of specific CTL. Third, the TsF-specific B16G monoclonal antibody was tested for its ability to modulate the effects of UV radiation in vivo. The i.v. injection of B16G into UV-irradiated mice reduced the suppression of CHS. Splenocytes of B16G-treated mice transferred into normal recipients, and they suppressed CHS, indicating that the Ts were not depleted. Fourth, B16G was used to isolate a putative TsF by antibody immunoadsorbance. When the B16G-bound fraction was eluted and injected i.v. into normal animals, it suppressed CHS and represented a 900-fold enrichment of activity over the starting material, based on specific activity. By SDS-PAGE, the B16G-bound material contained nondisulfide-linked 45- and 50-kDa components. These results suggest that TsF may play an immunoregulatory role in CHS. The isolation of a UV radiation-induced TsF lends credence to the involvement of such molecules. ^

Mechanisms of multidrug resistance: Differences between natural and acquired adriamycin -resistant human colon carcinoma cells

Mechanisms of multidrug resistance (MDR) were studied in two independent MDR sublines (AdR1.2 and SRA1.2) derived from the established human colon carcinoma cell line LoVo. AdR1.2 was developed by long-term continuous exposure of the cells to adriamycin (AdR) in stepwise increments of concentration, while SRA1.2 was selected by repetitive pulse treatments with AdR at a single concentration. In this dissertation, the hypothesis that the mechanism of drug resistance in SRA1.2 is different than that in AdR1.2 is tested. While SRA1.2 demonstrated similar biological characteristics when compared to the parental LoVo, AdR1.2 exhibited remarkable alterations in biological properties. The resistance phenotype of AdR1.2 was reversible when the cells were grown in the drug-free medium whereas SRA1.2 maintained its resistance for at least 10 months under similar conditions. Km and Vmax of carrier-mediated facilitated diffusion AdR transport were similar among the three lines. However, both resistant sublines exhibited an energy-dependent drug efflux. AdR1.2 appeared to possess an activated efflux pump, and a decreased nucleus-binding of AdR, whereas SRA1.2 showed merely a lower affinity in binding of AdR to the nuclei. Southern blot analysis showed no amplification of the MDR1 gene in either of the two resistant subclones. However, Western blot analysis using the C219 monoclonal antibody against P170 glycoprotein detected a Mr 150-kDa plasma protein (P150) in AdR1.2 but not in SRA1.2 or in the parental LoVo. In vitro phosphorylation studies revealed that P150 was a phosphoprotein; its phosphorylation was Mg$\sp{2+}$-dependent and could be enhanced by verapamil, an agent capable of increasing intracellular AdR accumulation in AdR1.2 cells. The phosphorylation studies also revealed elevated phosphorylation of a Mr 66-kDa plasma membrane protein that was detectable in the AdR1.2 revertant and in AdR1.2 when verapamil was present, suggesting that hyperphosphorylation of the Mr 66-kDa protein may be related to the reversal of MDR. SDS-PAGE of the plasma membrane protein demonstrated overproduction of a Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa protein was not immunoreactive with C219, but its absence in the AdR1.2 revertant and the parental LoVo suggests that it is an MDR-related plasma membrane protein. In conclusion, the results from this study support the author's hypothesis that the mechanisms responsible for "Acquired" and "Natural" MDR are not identical. ^