Calcium ions that are released from the Sarcoplasmic Reticulum (SR) upon electrical stimulation bind to troponin C of the thin filament structure, and initiate contraction of the skeletal muscle fiber. In order for the muscle fiber to relax, calcium must dissociate from troponin C and be removed from the cytoplasm by reuptake via the SR Calcium ATPase or by binding to cytoplasmic proteins. The intricacies of the calcium removal system in intact mammalian fibers have not been elucidated. The goal of my thesis project was to characterize the calcium removal system in mammalian intact skeletal muscle fibers, and determine the contribution of the individual components involved, such as the SR calcium ATPase, troponin C, and parvalbumin. Rat fast-twitch flexor digitorum brevis fibers (FDB) and slow-twitch soleus fibers are enzymatically dissociated and suspended in low melting temperature agarose gel to minimize fiber movement during fluorescence recordings. FDB fibers and soleus fibers are loaded with fura-2 (cell permeant form) and electrically stimulated by 1 to 40 pulses. Florescence signals are recorded at 380 (calcium sensitive) and 358 (calcium insensitive) nm excitation. Ca2+ is calculated assuming non-instantaneous equilibrium with fura-2. The rate constant of calcium decay decreased significantly with increasing stimulation duration in the FDB fibers, but remained relatively constant in the soleus fibers. This is due to expected differences in parvalbumin concentration between fast-twitch and slow-twitch fibers. In fast-twitch fibers parvalbumin becomes increasing saturated by calcium with increasing stimulation durations and can no longer contribute to the decay of calcium. However, there is negligible amounts of parvalbumin in slow-twitch fibers, therefore they do not exhibit this slowing of calcium decay effect. Quantification of the SR calcium ATPase, troponin C and parvalbumin content, using SDS page and immunoblotting techniques confirmed that there was a significant difference in the concentration of parvalbumin between rat FDB (1.2 mM calcium binding site concentration) and soleus fibers ({dollar}<{dollar}50 {dollar}\mu{dollar}M calcium binding site concentration). Unexpectedly there was no significant difference in the concentration of SR calcium ATPase, and troponin C. The values determined by the gel and immunoblot Quantification were well supported by preliminary modeling analysis of the Ca2+ decay. In conclusion, there are significant differences in the decay of Ca2+ in rat fast-twitch and slow-twitch muscle, which is due to differences in parvalbumin concentration. This indicates that parvalbumin has a significant role in the decay of calcium in mammalian skeletal muscle fibers.

The Chinese hamster lung cell line DC-3F8/A55 has a 4,500-fold increase in resistance to methotrexate over the parental cell line DC-3F. Although DC- 3F8/A55 cells have a 4.5-fold increase in a mutant form of dihydrofolate reductase, this does not fully account for the high level of resistance to methotrexate. The purpose of this study is to determine the molecular basis for the inability of DC-3F8/A55 cells to accumulate methotrexate, and to identify the mechanism of transport of folate compounds in DC-3F8/A55 cells. This work has revealed that DC-3F8/A55 cells harbor a debilitating mutation to the reduced folate carrier gene, resulting in the loss of reduced folate carrier function. A nonsense mutation changes an arginine at amino acid 88 to a STOP codon, resulting in a non-functional protein. The parental cell line DC-3F is heterozygous at this locus, possessing one mutant and one wild-type allele of the RFC gene, thus retaining reduced folate carrier activity. These facts are supported by the kinetics of folate transport in both of these cell lines. The parental cell line DC-3F has a Kt for folinic acid of 10.69 +/- 0.67 muM and for methotrexate of 8.88 +/- 0.82 muM, values characteristic of a cell expressing a reduced folate carrier. DC-3F8/A55 cells were found to have a Kd for MTX of 3.16 +/- 1.03 nM, for folinic acid of 7.75 +/- 2.16 nM, and for folic acid of 1.42 +/- 0.54 nM. The high affinity of DC-3F8/A55 cells for folic acid, with a Kd for folic acid in the nM range, suggests that these cells are expressing a folate receptor. Northern blot analysis revealed a 1.6 kb transcript with low homology to FR-alpha and FR-gamma in DC-3F8/A55 cells. Overall, these studies suggest that the methotrexate transport-defective cell line DC-3F8/A55 expresses a previously unidentified folate receptor which may be a new member of the folate receptor family.

Intracellular Ca2+ pools are essential elements in the generation of Ca2+ signals within cells, however, their nature and identity have remained elusive. Ca2+ pools are complex and dynamic entities and are modified by G protein-induced membrane fusion events, allowing GTP-activated transfer of Ca2+ between discrete Ca2+ pools. Studies examined the modification of GTP-activated Ca2+ translocation process by fatty acyl-CoA esters and fatty acids since G protein action can be modified by fatty acylation. Using permeabilized DDT1MF-2 smooth muscle cells, palmitoyl-CoA (IC50 = 0.5 muM) was observed to completely block 45Ca2+ release activated by GTP, while having no effect on InsP3-induced Ca2+ release. Fatty acyl chain length was important, only C-13 to C-16 fatty acyl-CoA esters fully inhibited the action of GTP. CoA(10 muM) also blocked GTP-activated Ca2+ release, although the free sulfhydryl group and ATP requirements indicated that CoA must be fatty acylated to be effective. The nonhydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, blocked the GTP effect identically to myristoyl- and palmitoyl-CoA. Thus, fatty acyl transfer is not required indicating that the blockade is due to a direct allosteric modification of a component of the GTP-activated process. Palmitoyl-CoA not only inhibited but completely reversed GTP-activated Ca2+ release. In the presence of oxalate, GTP-activated Ca2+ transfer causes a substantial increase in Ca2+ accumulation; palmitoyl-CoA also completely reversed this effect. These results provide strong evidence that GTP-activated Ca2+ translocation does not reflect a full fusion event, but the formation of a reversible prefusion pore. The actions of fatty acids were very different from their acyl-CoA esters; 10-100 muM palmitate (C16:0) had a major stimulatory effect on GTP-mediated Ca2+accumulation. The biphasic nature of the palmitate effect was characteristically similar to the effect of oxalate; however, the EC50 for palmitate was 20 muM (approx. 100-fold lower that of oxalate). This activation was highly specific for chain length and degree of saturation. Only pentadecanoic acid (C15:0) duplicated this effect, unsaturated fatty acids were completely ineffective. Both palmitate- and oxalate-activated Ca2+ accumulation in the presence of GTP were inhibited by the anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS). Hence, both Ca2+-complexing agents may enter anion-permeable Ca2+ subpools through similar anion channels. To further examine the nature of these Ca2+ complexes, a comparison of the releasability of Ca2+ using InsP3 and the Ca2+ ionophore, A23187 was undertaken. In the presence of oxalate, GTP-mediated accumulation of Ca2+ was only slowly releasable by InsP3 or A23187. Whereas, Ca2+ accumulated in the presence of palmitate and GTP was completely releasable by A23187, only a small fraction of the accumulated Ca2+was released by InsP3. These data suggest important differences between the state and possibly, the location of oxalate- and palmitate-Ca2+ complexes within Ca2+ pools. Thus, the formation of Ca2+-fatty acid complexes and, in turn, the activation of Ca2+ accumulation may reflect a major physiological role for fatty acids in stabilizing Ca2+ within the lumen of Ca2+ pools.

The inositol 1,4,5-trisphosphate (InsP{dollar}\sb3{dollar})-sensitive Ca{dollar}\sp{lcub}2+{rcub}{dollar} pools of {dollar}\rm DDT\sb1MF{lcub}-{rcub}2{dollar} smooth muscle cells empty after inhibition of the intracellular Ca{dollar}\sp{lcub}2+{rcub}{dollar}-ATPase by thapsigargin. Pool emptying causes cells to cease division and enter a stable, quiescent G{dollar}\sb0{dollar}-like state. High serum treatment of these pool-depleted quiescent cells induces the reappearance of functional Ca{dollar}\sp{lcub}2+{rcub}{dollar} pump protein, re-filling of InsP{dollar}\sb3{dollar}-sensitive Ca{dollar}\sp{lcub}2+{rcub}{dollar} pools, and re-entry of cells into the cell cycle. This recovery is mediated by the direct donation of essential fatty acids from the high serum to the pool-depleted cells. The essential fatty acids linolenic acid, linoleic acid, and arachidonic acid each induced recovery of Ca{dollar}\sp{lcub}2+{rcub}{dollar} pools and re-entry of cells into the cell cycle with an EC{dollar}\sb{lcub}50{rcub}{dollar} of approximately 5 {dollar}\mu{dollar}M; the action of each was dependent on protein synthesis. All non-essential fatty acids and growth factors tested did not promote recovery. Inhibitors of the prostanoid and lipoxygenase metabolism pathways had no effect on essential fatty acid-induced Ca{dollar}\sp{lcub}2+{rcub}{dollar} pool or growth recovery. However, the cytochrome P-450 inhibitors SKF-525A, metyrapone, and nordihydroguaiaretic acid each prevented the action of AA. Importantly, treatment of quiescent cells with either of 8,9- or 11,12-epoxyeicosatrienoic acid (EET), two of the four regiospecific cytochrome P-450 metabolites of arachidonic acid, also induced recovery. However, further metabolism of the effective EETs either to dihydroxyeicosatrienoic acid metabolites or through subsequent eicosanoid synthesizing pathways appears to be unnecessary. Evidence suggests that neither protein kinase activity nor the participation of cGMP or cAMP is involved in the EET-induced recovery process. These results are important in understanding the role of cytochrome P-450 derived eicosanoids in cellular regulation and the relationship between pool emptying and cell cycle control.

The mammalian cell DNA synthetic machinery has been isolated from human cervical carcinoma HeLa and murine FM3A cells, and characterized as a multiprotein replication complex (MRC). This complex is fully capable of supporting SV40 in vitro DNA replication in the presence of viral large T antigen. To further study the regulation of the activity of the MRC during cell differentiation, a human leukemia HL-60 cell model was used. HL-60 cells are able to undergo macrophage-like differentiation after exposure to a phorbol ester. The differentiation is accompanied by the cessation of DNA synthesis. In the attempt to isolate and characterize the HL-60 cell DNA synthetic machinery, we found that several essential DNA replication proteins such as DNA polymerases alpha and delta, DNA primase, replication factor C (RF-C), replication protein A (RP-A), proliferating cell nuclear antigen (PCNA), topoisomerases I and II form a multiprotein complex in HL-60 cells, which is consistent with the finding in HeLa and FM3A cells. The DNA synthetic machinery is therefore believed to be commonly present in mammalian cells as an organized protein complex, and is designated the DNA synthesome. The DNA synthesome is absent from HL-60 cells induced to differentiate by TPA. Western blot analysis showed that TPA-induced differentiation is accompanied by the disassembly of the DNA synthesome, which is consistent with the observed cessation of DNA synthesis. RNA analysis suggested that the disassembly of the DNA synthesome is regulated at the gene expression level. Studies of the integrity and activity of the DNA synthesome in aphidicolin-arrested HL-60 cells showed that the synthesome is present in the temporarily arrested cells but in an inactive form. Taken together, these evidence suggested that the disassembly of the DNA synthesome is a differentiation-specific cellular event, and that the biochemical status of the DNA synthesome can distinguish the difference between temporary and permanent cell growth arrest.

Lipids are amphiphilic molecules that are major components of biological membranes. In an aqueous environment, they typically form ordered, bilayer structures. However, some naturally occurring lipids, when isolated and hydrated under physiologic conditions, will preferentially form inverted, non-bilayer structures, e.g., the hexagonal H{dollar}\sb{lcub}\rm II{rcub}{dollar} phase which consists of lipid cylinders with the polar head groups facing in lining an aqueous core. When incorporated into a bilayer, such polymorphic lipids impart considerable intramembrane stress. This dissertation describes research carried out to test the hypothesis that polymorphic lipids play a key role in modulating the behavior of integral membrane proteins. Biochemically active membranes such as the energy-transducing membranes of mitochondria and chloroplasts are highly enriched in polymorphic lipids. The adenine nucleotide translocator (AdNT), an integral protein of the mitochondrial inner membrane (i.m.) which catalyzes the exchange of ADP for ATP across the i.m., has been isolated and reconstituted into liposomes. The polymorphic lipid content of the reconstituted system was systematically varied, and the effects of this variation on translocator function were determined. The mitochondrial AdNT was successfully reconstituted using a modification of existing protocols. The reconstituted system was characterized and found to have properties consistent with those previously reported for both in situ and reconstituted AdNT. A method was developed for the determination of translocator specific activity in the reconstituted system based on tight-binding inhibitor theory. Reconstituted AdNT activity was found to be a smooth function of polymorphic lipid content, with a maximum at a lipid composition similar to that of the i.m. Liposome leakiness increased with increasing polymorphic lipid content. No significant effect of polymorphic lipid content on either extent of AdNT incorporation or AdNT orientation was detected.

A Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase, a membranous protein in sarcoplasmic reticulum (SR) of skeletal muscle, is an enzyme that catalyzes transport of cytoplasmic Ca{dollar}\sp{lcub}2+{rcub}{dollar} into the SR lumen upon ATP hydrolysis. The endoplasmic reticulum (ER) Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase from rat liver cells, but not SR Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPases from skeletal or cardiac cells, was inhibited by thapsigargin, a sesquiterpene lactone from an umbelliferous plant Thapsia garganica. In this thesis project, thapsigargin (TG) was shown to be a potent inhibitor of all the SR/ER-type Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPases, but not the plasma membrane Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase in erythrocytes or the ryanodine receptor in skeletal muscle. TG inhibition is stoichiometric and potent: the affinity of TG to the Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase is high with the dissociation constant in subnanomolar. TG binding to the ATPase is apparently irreversible, even though the interaction is non-covalent. TG inhibits the steady state activities, Ca{dollar}\sp{lcub}2+{rcub}{dollar} transport and ATP hydrolysis of the SR Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase, as well as partial activities of the ATPase: Ca{dollar}\sp{lcub}2+{rcub}{dollar} and ATP binding at equilibrium and phosphoenzyme formation from inorganic phosphate are equally affected. Kinetic studies revealed that TG tightly binds an ATPase intermediate, which is produced during the ATPase catalytic cycle. The ATPase conformation (intermediate) sensitive to TG interaction can be also produced by the removal of Ca{dollar}\sp{lcub}2+{rcub}{dollar} by EGTA. The complex of TG and the ATPase intermediate does not react with any ATPase ligand and, therefore, is a dead-end complex. Spectroscopic and biochemical studies showed that this dead-end complex is in a different conformational state from the original intermediate that TG is initially binding. The changes in the ATPase conformation upon TG binding may be responsible for the global inhibition of ATPase partial activities. Since these partial activity sites (the nucleotide binding site, Ca{dollar}\sp{lcub}2+{rcub}{dollar} binding site, and the phosphorylation site) affected by TG are distantly located in the ATPase, the potent and stoichiometric inhibition of the ER/SR-type Ca{dollar}\sp{lcub}2+{rcub}{dollar} ATPase by TG may occur at either a location that is critical in the function of all the partial activities of the ATPase or a location essential for transduction of the signals between the sites. Determination of the TG interaction site showed that TG is not binding to the phosphorylation site, the Ca{dollar}\sp{lcub}2+{rcub}{dollar} binding site, or the nucleotide binding site. (Abstract shortened with permission of author.)

The purpose of this project was to identify a rich source of Type II estrogen receptor (Type II ER), purify the receptor to homogeneity and determine its physical and pharmacological properties. Our results showed that pregnant rat uterus is highly enriched in the Type II ER; therefore this tissue has been used to study this receptor. The investigation demonstrated that the crude low affinity Type II ER (Kd {dollar}\sim{dollar}30nM) is significantly elevated during pregnancy and becomes the major {dollar}(\ge{dollar}88%) form of ER in rat uteri, while the high affinity Type I estrogen receptor (Type I ER, K{dollar}\sb{lcub}\rm D{rcub}{dollar} {dollar}\sim{dollar}0.10nM) form remained unchanged. This result may suggest a possible role for the low affinity ER in pregnancy. The Type II ER has been purified to homogeneity from uteri obtained from pregnant rats. The purified receptor {dollar}(>{dollar}97% purity) has an apparent estradiol binding affinity of 24 nM with a Bmax of 13 nmol/mg protein. HPLC analysis indicates that the purified Type II receptor has a native molecular weight of 63 kDa while the denatured molecular weight on SDS-PAGE is about 58 kDa. The purified receptor has a steroid specificity for estradiol {dollar}\ge{dollar} quercetin {dollar}\ge{dollar} diethylstilbestrol {dollar}\ge{dollar} tamoxifen, = dihydrotestosterone. The receptor appears not to contain any DNA binding activity and lacks sigmoidal steroid binding activity. However the steroid binding of the Type II ER is sensitive to sulfhydryl group reagents. This study has also identified a ERE DNA binding activity that copurified with Type II ER during the initial stages of purification. However, upon further purification by ion exchange chromatography the DNA binding activity (ERE Binding) was separated from the Type II ER. This ERE binding protein eluted from a Q-sepharose column at a conductivity corresponding to 250 mM NaCl. The ERE binding activity appears to be a novel protein in that it formed a discrete, rapidly migrating complex with the ERE oligonucleotide. In contrast the high affinity Type I ER formed a heterogeneous mixture of multiple ER-ERE complexes. Immunological analysis demonstrate that the purified low affinity Type II ER was not recognized by antibodies directed against the DNA binding domain of the recombinant Type I ER or by anti-tyrosinase antibody. The results indicate that the Type II ER shares no sequence homology with tyrosinase or the Type I ER.

Increased matrix-Ca{dollar}\sp{lcub}2+{rcub}{dollar}, in combination with a triggering agent/condition which may be oxidative stress, increased temperature or one of a large number of chemicals, induces the inner mitochondrial membrane to undergo a permeability transition allowing entry/efflux of small {dollar}(<{dollar}1500 dalton) solutes. The transition is associated with a cluster of Ca{dollar}\sp{lcub}2+{rcub}{dollar}-dependent events including mitochondrial swelling, uncoupling, collapse of the membrane potential, oxidation of pyridine nucleotides, and a transition from the aggregated to orthodox state. Although the transition has been implicated in hypoxia/reperfusion injury, heavy metal toxicity, Reyes syndrome, and the toxicity of redox active drugs and xenobiotics and is proposed to mediate non-shivering thermogenesis, its mechanistic details have yet to be clearly elucidated. The objective of this thesis was to clarify the biochemical process(es) underlying the mitochondrial permeability transition. We have identified spermine as a novel inhibitor of the transition in isolated mitochondria from rat heart and liver. The transition was induced by a series of triggering agents, t-butyl hydroperoxide (t-BH), Ca{dollar}\sp{lcub}2+{rcub},{dollar} carboxyatractylate, phenylarsine oxide and elevated inorganic phosphate (P{dollar}\sb{lcub}\rm i{rcub}){dollar} concentrations. Regardless of triggering agent, spermine inhibited three phenomena diagnostic for the transition: Ca{dollar}\sp{lcub}2+{rcub}{dollar}-release, mitochondrial swelling and pyridine nucleotide oxidation. Concentrations of spermine producing inhibition fell well within the physiological range. Spermine was then used together with the series of triggering agents under well defined conditions, to delineate more precisely the mechanism(s) by which spermine and selected other triggering agents and inhibitors modulate transition occurrence. Our results indicate that (1) Spermine inhibition is highly sensitive to the ionic composition of the medium; K{dollar}\sp+{dollar} antagonizes spermine action. (2) K{dollar}\sp+{dollar} and spermine act on sites external to the mitochondria. (3) The triggering agent P{dollar}\sb{lcub}\rm i{rcub}{dollar} induces the transition by decreasing the matrix concentration of ADP, an internal inhibitor of the transition. (4) Spermine inhibits the transition by enhancing the efficacy of ADP, most probably by increasing the affinity of the ADP binding site. (5) Ca{dollar}\sp{lcub}2+{rcub}{dollar} and t-BH induce the transition via mechanisms which are clearly distinct from the triggering action of P{dollar}\sb{lcub}\rm i{rcub}.{dollar}