Peter S. Coleman

Membrane cholesterol, tumorigenesis, and the biochemical phenotype of neoplasia.

The loss of sensitivity to feedback control of lipid synthesis in tumors is especially manifest with regard to cholesterol biosynthesis. This phenomenon has been documented in a wide variety of cells soon after exposure to a carcinogenic insult, and results in the progressive enrichment of diverse cellular membranes with cholesterol during the tumorigenic process. By the time a tumor is identifiable histopathologically, such membrane enrichment with cholesterol can lead to significant changes in the activities of membrane-affiliated enzymes. This, in turn, can effect a wide variety of altered metabolic patterns within the afflicted cell, thereby promoting altered cell function. Some of the altered metabolic patterns in tumors that are consistent with events leading to cholesterol-enriched membranes include increased glutamate and glutamine oxidation in support of tumor bioenergetics; reduced rates of glucose oxidation via respiration together with enhanced levels of aerobic glycolysis; diminished urea cyc...

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR, diffusion and entrapment analyses.

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1--0.4 micron with a mean diameter of 0.21 micron.

Enhancement of carbonic anhydrase activity by erythrocyte membranes.

The human erythrocyte membrane is an efficient enhancer of both high (CA II) and low (CA I) activity isozymes of red blood cell carbonic anhydrase. The presence of membrane increased CO2 hydration catalyzed by bovine CA II 1.6-fold, human CA II 3.5-fold, and human CA I 1.6-fold. With the high activity CA isozymes, maximal stimulation was observed in the presence of 1-3 micrograms membrane protein/ml. The Vmax for bovine CA II (4 nM) rose from 0.302 to 0.839 mM/s, while that for human CA II (6 nM) increased from 0.113 to 0.414 mM/s in the absence and presence of membrane, respectively. The apparent Km for CO2 increased from 13.2 to 51.2 mM for bovine CA II, and from 6.5 to 38.5 mM for human CA II. Mixtures of membrane plus enzyme, upon centrifugation through linear sucrose density gradients, displayed enhanced Ca activity only in membrane-containing gradient fractions, verifying the stimulatory ability of membranes on enzyme activity and indicating tight and stable complex formation. Membrane enhancement of CA activity appears to be a general phenomenon in that mouse hepatocyte membranes also stimulated CA activity, although less efficiently than erythrocyte membranes. Of the many soluble putative effectors assayed, only imidazole enhanced CA II activity to an extent comparable with erythrocyte membranes; imidazole did not, however, stimulate the activity of human CA I. The data are consistent with a model of CA II activation by membrane association that may effect a distortion of the enzyme conformation in such a way as to facilitate intra- and/or intermolecular proton transfer between membrane-bound and enzyme-bound proton shuttling residues (perhaps the imidazole moiety of histidine) and the Zn-bound hydroxide at the catalytic site of the enzyme.

Cellular distribution of cholesterogenesis-linked, phosphoisoprenylated proteins in proliferating cells.

A set of isoprenylated proteins has been detected in rapidly proliferating, suspension‐grown murine lymphoma cells. Our evidence indicates that all of these isoprenylated proteins are phosphorylated. Subsequent to a 24 h incubation with mevinolin to deplete the intracellular mevalonate (MVA) level, cells were incubated with [3H]MVA and/or 32Pi and both total cell and subcellular fraction proteins were resolved via 1‐ and 2‐D gel electrophoresis, then assessed via subsequent autoradiography. The phospho‐isoprenylated proteins comprise a set spanning a molecular mass range of 21–69 kDa and all dispay acidic pI. MVA‐derivatized proteins of 21–24 kDa, which consist of multiple isoforms, are present in both cytosolic and nuclear fractions. Larger phospho‐isoprenylated protein species (44–69 kDa) are specifically localized within the nucleus, where applicable extraction protocols indicate that they are part of or closely affiliated with the nuclear matrix‐intermediate filament (NM‐IF) components. The localization of the 69 kDa prenylated species within the NM‐IF fraction, together with evidence of its phosphorylation, supports recent indications that this protein is the nuclear matrix component lamin B.

Cholesterol enrichment of normal mitochondria invitro: A model system with properties of hepatoma mitochondria

Abstract It is known that rat hepatoma mitochondria contain higher levels of endogenous cholesterol than do mitochondria from normal liver. In the experiments described here, normal liver mitochondria were enriched with cholesterol by a solid-state transfer process in vitro and some of their enzymic properties were compared with literature values reported for hepatoma mitochondria. Striking parallels were seen. The data indicate that normal mitochondria, enriched with cholesterol in vitro , may create an interesting model system for examining some metabolic characteristics of tumor mitochondria.

Interactions of immunoglobulins with liposomes: An ESR and diffusion study demonstrating protection by hydrocortisone

Abstract Heat-aggregated IgG, used as a surrogate for immunoglobulin configuration in immune complexes, mobilized a hydrophobic spin probe within bilayers of anionic liposomes, whereas native IgG was ineffective. Hydrocortisone, preincorporated into liposomal bilayers, both prevented the perturbation of nonpolar membrane regions and reduced the accelerated rate of solute diffusion from liposomes provoked by aggregated immunoglobulin. The capacity of aggregated, but not native, IgG to initiate membrane responses of living cells may therefore be due to its ability to alter surface bilayers or similar configurations of plasma membrane Fc receptors, events antagonized by corticosteroids.

The role of lipid metabolism in neoplastic differentiation

Abstract Many structural and functional differences between normal and neoplastic tissues are known. However, any metabolic alteration proposed to be fundamental to the neoplastic process must (1) occur soon after the carcinogenic insult in a wide variety of tissues, and (2) generate other biochemical properties characteristic of tumor cell metabolism. Loss of feedback control of lipid synthesis has been shown to fulfill the first criterion. Here we show how this change also meets the second requirement by leading to other known features of tumor metabolism: increased glutamate and glutamine oxidation, enhanced aerobic glycolysis, altered amino acid levels in host body fluids, decreased urea cycle activity, increased polyamine synthesis, alterations in reducing equivalent shuttle activities, and alterations in cell membranes.

Uncoupling of oxidative phosphorylation by a stable free radical and its diamagnetic homolog

Abstract The stable free radical 1,1′-diphenyl-2-picrylhydrazyl and its hydrazine homolog are very potent uncouplers of oxidative phosphorylation in whole mitochondria. These molecules exhibit many of the same effects on respiratory kinetics and ATPase as does 2,4-dinitrophenol, but at low concentrations near 10 −7 M, and appear to act at a mechanistic locus common to other “true” uncouplers of oxidative phosphorylation.

Mitochondrial ATPase activity and AdN translocation with ε-ATP as substrate

Rat liver mitochondria display two types of ATPase activities: a low level Mg-stimulated ATPase activity which appears to be located within the intermembrane space and is not directly associated with oxidative phosphorylation [l-4] and a DNP-stimulated* FrATPase activity that is located on the inner surface of the inner membrane and is associated with the energy conservation process [4-g]. While the Mgstimulated ATPase activity has not been well characterized, the Fi-ATPase that is associated with oxidative phosphorylation has been studied extensively [6,8]. Prior to interaction with the latter enzyme, AdN added to a mitochondrial suspension must first traverse the outer mitochondrial membrane and intermembrane space and then be translocated into and possibly through the inner membrane by the ATP/ADP facilitative exchange carrier [4,9-l l] . This AdN translocase maintains a strict specificity for an intact adenine base and at least two phosphate groups [ 121. Thus, inosine, quanosine, and uridine nucleotides are not exchanged to appreciable extents [4,11]. However, it has been shown that the l-Noxide analog of ATP can be translocated approximately 30% as well as ATP [ 131. e-ATP (1 j@-ethenoadenosine 5’-triphosphate), a fluorescent analog of ATP [ 141, has been found to be a good replacement for ATP as a substrate for several soluble enzymes, but not for the chloroplast

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with ϵ-ATP

Abstract 1. The use of 1, N 6 -ethenoadenosine 5′-triphosphate (ϵ-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied. 2. Direct [ 3 H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ϵ-[ 3 H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F 1 -ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ϵ-ATP is hydrolyzed by a Mg 2+ -dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria. 3. ϵ-ATP can be utilized quite well by the exposed F 1 -ATPase of sonic submitochondrial particles. This ϵ-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F 1 -ATPase displays similar K m values for both ATP and ϵ-ATP; however, the V with ATP is approximately six times greater than with ϵ-ATP. 4. Since ϵ-ATP is a capable substrate for the submitochondrial particle F 1 -ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F 1 -ATPase complex, and its response to various modifiers of oxidative phosphorylation.