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Biomedical subjects

M Fry

Publications and source records attributed to M Fry.

At least 91 records · Page 5Linked to original sources
PubMed1981

Cardiolipin requirement for electron transfer in complex I and III of the mitochondrial respiratory chain.

Almost complete phospholipid depletion has been achieved for Complex I and III of the mitochondrial respiratory chain using a technique that involves elution on Sephadex LH-20 in the presence of Triton X-100. Enzymic activity may be regenerated by replenishment with phospholipid. However, restoration of enzymic activity in phospholipid-depleted Complex I and III has been shown to require the presence of cardiolipin. These results are, therefore, similar to findings on the absolute catalytic requirement of cardiolipin for cytochrome oxidase activity (Fry, M., and Green, D. E. (1980) Biochem. Biophys. Res. Commun. 93, 1238-1246). At least two roles for phospholipid involvement in electron transfer processes are proposed, a catalytic role provided specifically by cardiolipin and a dispersive role that may be provided by various phospholipids or detergents. The absolute requirement of enzymic activity for cardiolipin suggests that this phospholipid plays a crucial role in the coupled electron transfer process.

Animals↗
PubMed1981

On the activity and fidelity of chromatin-associated hepatic DNA polymerase-beta in aging murine species of different life spans.

Activity and accuracy of chromatin-directed DNA replication have been compared in young and aged Mus musculus and Peromyscus leucopus, two murine species with contrasting maximum lifespans. Chromatin isolated from livers of mature adults of both species copied efficiently exogenous DNA templates using predominantly DNA polymerase-beta. The DNA synthetic activity of liver chromatin remained constant in both species throughout their lifetimes. The fidelity of chromatin-directed poly [d(A-T)] synthesis was similar for the comparatively short-lived M. musculus and the relatively long-lived P. leucopus and remained unaltered in old animals. The fidelity of poly [d(A-T)] copying catalyzed by DNA polymerase-beta-dissociated from liver chromatin was comparable to that of the chromatin-directed synthesis. The dissociation enzymes did not exhibit diminished fidelity of poly [d(A-T)] synthesis with age. In all ages of both species examined, the murine liver DNA polymerase-beta, both chromatin-associated and solubilized, exhibited high error frequencies; approximately one dGMP was incorporated for every 500-1,000 complementary nucleotides polymerized. The relationship of these results to the accuracy of DNA replication and repair as a determinant of aging is considered.

Aging↗
PubMed1981

Studies on the resolution of cytochrome oxidase.

Cytochrome oxidase has been resolved in acetic acid and high salt/detergent media. In 0.5% acetic acid, the smaller subunits of the enzyme are selectively extracted with retention of an insoluble protein fraction containing subunits I-IV, VII. This fraction retains all the heme and copper of the original enzyme in a spectrally unaltered state, and possesses enzymic activity comparable to the unresolved enzyme. The further removal of subunit IV from this fraction results in migration of heme and copper and modification of their spectral characteristics. Resolution of the enzyme in a high salt/detergent medium extracts smaller subunits (V-VII) together with subunit IV and some heme and copper. The heme associated with this enzymically active extract has spectral characteristics that are partially suggestive of heme a3. It is suggested that the fraction of subunits I-IV,VII, resolved in dilute acetic acid, may represent the limit of resolution of the cytochrome oxidase complex that remains actively and spectrally indistinguishable from the original enzyme.

Acetates↗
PubMed1980

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta.

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.

Animals↗
PubMed1980

The localization of tightly bound cardiolipin in cytochrome oxidase.

One to two molecules of tightly bound cardiolipin are associated with resolved fractions of cytochrome oxidase containing subunits I to III or I to IV. Large scale isolation of subunits I to IV indicates the presence of approximately 0.5 molecule of cardiolipin per molecule of subunit I. Lipoprotein staining of sodium dodecyl sulfate/urea/acrylamide gels of cytochrome oxidase support the findings that subunit I is a lipoprotein. The resistance of this tightly bound cardiolipin to organic solvent extraction suggests a specific association of some tenacity with the protein.

Binding Sites↗
PubMed1980

Phospholipids as the molecular instruments of ion and solute transport in biological membranes.

Partition studies have established that phospholipids generally have the capabilities to mediate the transmembrane transport of the full range of ions and solutes that physiologically cross biological membranes. The list of transportable species includes cations, anions, amino acids, citric acid cycle intermediates, nucleotides, and sugars. Phospholipid-mediated transport can be readily modulated by altering the phospholipid mixture or by addition of detergents, nucleotides, divalent metals, proteins, peptides, or ring compounds. Containment of phospholipid within channels in protein appears to be the precondition for the formation of the micellar structure requisite for solute transport. Phospholipid-mediated transport is postulated to be a central feature of energy coupling, membrane-spanning systems, and membrane-bound, phospholipid-requiring enzymes.

Biological Transport↗
PubMed1980

Relation between enzymic catalysis and energy coupling.

The principles that underlie enzyme catalysis also apply to energy coupling processes. A comparison is made between a kinase system that mediates the phosphorylation of glucose by ATP (hexokinase), as the prototype for enzymic catalysis, and the mitochondrial electron-transfer complexes, as the prototypes for energy coupling systems. Induced polarization of chemical bonds and charge separation and elimination are common component events of both enzyme catalysis and energy coupling. Thus, definite limits can be imposed on models of energy coupling; they must comply with the basic principles of enzymic catalysis.

Binding Sites↗
PubMed1980

Ion-transport chain of cytochrome oxidase: the two chain-direct coupling principle of energy coupling.

Cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) couples the aerobic oxidation of ferrocytochrome c to the cyclical transport of monovalent cations or to the active transport of monovalent and divalent cations. This transport capability is mediated by an intracomplex ion-transport chain of two protein-bound molecules of cardiolipin per molecule of cytochrome oxidase. Cardiolipin in a two-phase system shows the identical ionophoric pattern as does the cytochrome oxidase coupled system. A molecular model of the cardiolipin chain suggests the possibility of a cage-like structure through which cations can be transferred from phosphate group to phosphate group. The ion-transport chain and the electron-transport chain are anchored to the same set of subunits (I+IV); the close proximity of the two chains argues for the direct coupling of electron and cation flow. The ion-transport chain of cytochrome oxidase provides an introduction to the molecular mechanisms by which ions are moved across membranes in energy-coupling systems.

Amino Acids↗
PubMed1980

On reagents that convert cytochrome oxidase from an inactive to an active coupling state.

Cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) of beef heart mitochondria, prepared by a standard method and brought to the highest purity level, is essentially inactive when tested in the aerobic assay involving oxidation of reduced cytochrome c by molecular oxygen. Three reagents (lysolecithin, Tween 20, and exogenous phospholipids) can convert cytochrome oxidase from an inactive to an active coupling state. These conversions are reversible: i.e., removal of the inducing agent leads to loss of activity. The evidence for the intrinsic coupling capability is that cytochrome oxidase in the active state invariably generates a proton gradient during respiration, and such gradient formation is demonstrable even when cytochrome oxidase is not inserted into a liposome.

Animals↗
PubMed1979

Cell adhesion and acquisition of detergent resistance by the cytoskeleton of cultured chick fibroblasts.

About 30% of the proteins of adherent cultured chick embryo fibroblasts are not solubilized by the non-ionic detergent Triton X-100 and remain firmly attached to the substratum. The insoluble residue contains a considerable part of the cell's cytoskeleton and its major constituents are large external transformation-sensitive (LETS) protein, the heavy chain of myosin, a 52,000 molecular weight protein and actin. Kinetic studies reveal that cytoskeleton insolubility in Triton is acquired either concurrently with cell adhesion or very closely with it. Neither cell adhesion nor binding of the Triton cytoskeleton to the substratum require de novo synthesis of protein. In the attempt to assess the role of LETS protein in cytoskeleton attachment, we find that trypsin-detached cells rapidly acquire Triton-insoluble cytoskeleton although their LETS protein content is about 15--20% of its level in long-term cultures. Removal of the great majority of LETS molecules of adherent cultures by either urea or trypsin treatment does not affect the relative amount or composition of the anchored cytoskeletal proteins. Also, LETS protein of cultures exposed to cycloheximide for extended periods of time, is reduced to 10% of its maximum amount without much affecting the attachment and composition of the cytoskeleton. It is deduced that the great majority of LETS protein is not required for the attachment of the Triton cytoskeleton to the substratum.

Actins↗